Studies of the S-Adenosyl-L-Methionine Methyltransferases from Suspension Cultures of Ailanthus Altissima (Mill.) Swingle (Simaroubaceae)

STUDIES OF THE S-ADENOSYL-L-METHIONINE METHYLTRANSFERASES FROM SUSPENSION CULTURES OF AILANTHUS ALTISSIMA (MILL.) SWINGLE (SIMAROUBACEAE)

OLUBUKOLA AFOLUSHO OSOBA

A thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy.

Department of Pharmacognosy The School of Pharmacy University of London 1994 ProQuest Number: 10104884

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor Dr. M.F. Roberts and her husband Ltc. B.C. Homeyer (AUS, Rtd.), for their guidance, constructive criticism and assistance. Their contribution was vital to the successful completion of this thesis. I also wish to thank the Organisation of African Unity (STRC), for the award of the main grant, which made these studies possible. I am also grateful to the African Educational Trust and the Family Association for the awards of additional augmenting grants. I am grateful to all personnel who supplied various essential services: the Mass Spectroscopy Unit of the School of Pharmacy and the NMR unit of the King’s College, University of London for running mass and NMR spectra; the media kitchen of the School of Pharmacy for sterilisation services; Yang Shi-Lin for supplying some enzyme substrates; Annie Cavanagh for the photography and diagrams; Maureen Pickett and Gus Ronngren for supplying laboratory equipment and chemicals and Ron Cooper for washing up the glassware. I hereby applaud the members of the Pharmacognosy department for their genuine friendship and kindness, which made life in the School of Pharmacy extremely pleasant. I would also like to acknowledge certain friends including: Nike, Ronke, Lolade, Caroline, Yinka, Angela, Thi and Dorothy, for their support during the difficult times when my grant ran out, and for their assistance with typing and proof reading my thesis. I am also grateful to members of my immediate family which include: my parents, my brother, Leke and sister-in-law, Yinka and my sisters Yinky and Bunmi, for their steadfast support, encouragement and financial assistance, as well as my nephew and niece, Shubby and Toni, for the laughter and fun times we shared. Finally, I wish to express my profound gratitude to my Heavenly Father, my Lord Jesus and the Holy Ghost, as I acknowledge their supernatural intervention in granting me the presence of mind, and the strength I needed to complete my thesis, which is dedicated firstly to God’s glory and secondly to the memory of my granny ‘Olobi’. TABLE OF CONTENTS Title Page 1 Acknowledgements 2 Table of Contents 3 List of Figures 15 List of Tables 18 List of Abbreviations 19 Abstract 22

1. INTRODUCTION 24 1.1 Studies of Ailanthus altissima 25 1.1.1 Ailanthus altissima as a Plant 25 1.1.1.1 A. altissima Tree 25 1.1.1.2 A. altissima Cultures 27 1.1.2 The Uses of A. altissima in Traditional Medicine 27 1.1.3 Secondary Metabolites from the Intact Plant of A. altissima 29 1.1.3.1 Alkaloids 29 1.1.3.2 Quassinoids 29 1.1.3.3 Quinones 29 1.1.4 Secondary Metabolites form A. altissima Cell Cultures 30 1.1.4.1 Alkaloid Production 30 1.1.4.2 Quassinoid Production 38 1.1.4.3 Coumarin Production 39 1.1.5 Studies on the Biological Activities of the Quassinoids and Canthin- 6-one Alkaloids of A. altissima 39 1.1.5.1 Quassinoids 39 1.1.5.2 Alkaloids 40 1.2 Plant cell cultures as Alternatives to Intact Plants 43 1.2.1 The Commercial Value of Plants in the Pharmaceutical Industry 43 1.2.2 The Application of Plant Cell Cultures in Drug Production 44 1.2.2.1 Advantages of Plant Cell Cultures in Secondary Metabolite Production 45 1.2.2.2 The Commercial Production of Drugs and Secondary Metabolites by Plant Cell Cultures 46 1.2.3 Plant Cell Cultures in Biotransformation Reactions 48 1.2.3.1 Formation of Cardenolides in Digitalis lanata Cell Cultures 49 1.2.3.2 Biosynthesis of Vinblastine and Vincristine in Plant Cell Cultures by Biotransformation 49 1.2.4 Plant Cell Cultures in the Study of Biosynthetic Pathways 50 1.2.4.1 Plant Cell Cultures in the Study of the Biosynthesis of Canthin- 6-one Alkaloids 52 1.2.4.1.1 The Role of Tryptophan and Tryptamine in the Biosynthesis of Canthin- 6-one Alkaloids 53 1.2.4.1.2 The Role of a-Ketoglutarate in the Biosynthesis of Canthin- 6-one Alkaloids 58 1.2.4.1.3 The Methylating Ability of A. altissima Cell Suspension Cultures 60 1.2.4.2 Plant Cell Cultures in the Study of Coumarin Biosynthesis 61 1.2.4.2.1 Biosynthesis of Coumarins from L-Phenylalanine 62 1.2.4.2.2 Biosynthesis of Coumarins Lacking Oxygenation at the 7-Position 63 1.2.4.2.3 Biosynthesis of 7-Oxygenated Coumarins 65 1.2.4.2.4 Biosynthesis of di- and tri- Hydroxycoumarins 67 1.2.4.2.5 Biosynthesis of Methylated Coumarins: Scopoletin, Fraxetin, Isofraxidin and Puberulin 68 1.3 Methyltransferases in Plants 70 1.3.1 O-Methyltransferases in Plants 72 1.3.1.1 O-Methylation of Caffeic Acid in Lignin and Flavonoid Biosynthesis 72 1.3.1.2 O-Methylation of Flavonoid Compounds 73 1.3.1.2.1 4'-0-Methylation of Isoflavones in Isoflavone Biosynthesis 73 1.3.1.2.2 5-0-Methylation of Isoflavones 74 1.3.1.2.3 O-Methylation of (+) 6a-Hydroxy- maackiain in Pisatin Biosynthesis 76 1.3.1.2.4 3'-0-Methylation of Luteolin in Flavone Glucoside Biosynthesis 76 1.3.1.2.5 7-O-Methylation of 3-Methyl- quercetin in Flavone Glucoside Biosynthesis 77 1.3.1.3 5 -0 -and 8 -O-Methylation of Furanocoumarins 78 1.3.1.4 3-O-Methylation of Dopamine in the Biosynthesis of Mescaline 79 1.3.1.5 4'-0-Methylation of 3'-Hydroxy-N-Methyl-(S)- coclaurine in the Biosynthesis of Retieuline 80 1.3.2 N-Methyltransferases in Plants 83 1.3.2.1 N-Methylation of the Amine, Putrescine, to N-Methylputrescine in the Biosynthesis of Tropane and Pyridine Alkaloids 83 1.3.2.2 N-Methylation of Nicotinic Acid 84 1.3.2.3 N-Methylation of Anthranilic Acid 85 1.3.2.4 N-Methylation of Xanthines to yield Caffeine 85 1.3.2.5 N-Methylation of Alkaloids 87 1.3.2.5.1 N-Methylation of Coniine to N-Methylconiine 87 1.3.2.5.2 N-Methylation of Cytisine in Quinolizidine Alkaloid Biosynthesis 87 1.3.2.5.3 N-Methylation of Hydroxytabersonine in Indole Alkaloid Biosynthesis 88 1.3.2.5.4 N-Methylation of (S)-Coclaurine in the Biosynthesis of Reticuline 89 1.3.2.5.5 N-Methylation of Tetrahydroproto- berberine Alkaloids in the Benzylisoquinoline Alkaloid Biosynthesis 90 1.4 Aims and Objectives of the Present Study of Ailanthus altissima 93 2. MATERIALS AND EXPERIMENTAL METHODS 94 2.1 Development of Ailanthus altissima cultures 95 2.1.1 Plant material 95 2.1.2 Callus Formation 95 2.1.3 Development of Suspension Cultures 95 2.1.4 Preparation of Growth Media 96 2.1.4.1 Murashige and Skoog Liquid Growth Medium 96 2.1.4.2 Murashige and Skoog Solid Growth Medium 96 2.2 Phytochemical Investigations ofAilanthus altissima cultures 97 2.2.1 Extraction of Secondary Metabolites from Tissue Cultures of A. altissima 97 2.2.2 Qualitative and Quantitative Identification of Secondary Metabolites from A. altissima cultures 97 2.2.2.1 Identification of Alkaloids and Coumarins in A. altissima cell cultures by Thin Layer Chromatography (TLC) 98 2.2.2.1.1 Solvent Systems Used for TLC of Alkaloid Reference Compounds 98 2.2.2.1.2 Solvent System Used for TLC of Coumarin Reference Compounds 99 2.2.2.2 Identification and Quantification of Alkaloids and Coumarins of A. altissima by High Performance Liquid Chromatography (HPLC) 99 2.2.2.2.1 Identification of Alkaloids of A. altissima by HPLC 99 2,22.22 Quantification of Alkaloids of A. altissima by HPLC 100 2.2.2.2.3 Identification of Coumarins of A. altissima by HPLC 100 2.2.2.2.4 Quantification of Coumarins of A. altissima by HPLC 101 2.2.2.3 Spectroscopic Methods for the Qualitative Identification of Secondary Metabolites of A. altissima 101 2.2.2.3.1 Fluorescence Spectroscopy 101 2.2.2.3.2 Ultraviolet Spectroscopy 102 2.2.2.3.3 Mass Spectroscopy 102 2.2.2.3.4 Proton-Nuclear Magnetic Resonance Spectroscopy 102 2.3 Methods Used for the Extraction, Isolation and Purification of Methyltransferases from A. altissima 103 2.3.1 Preparation of Buffers 103 2.3.1.1 Homogenisation Buffer (A) 103 2.3.1.2 Running Buffer (B) 103 2.3.1.3 Buffers for the Determination of Optimum pH (C) 105 2.3.2 Enzyme Extraction from A. altissima Cell Suspension Cultures 107 2.3.3 Assay for Canthin- 6-one Alkaloid and Coumarin Methyltransferase Activities in Cell Suspension CnXXuxQS of A. altissima 107 2.3.3.1 Preparation of the Incubation Mix and its Constituents 108 2.3.3.1.1 Preparation of Hydroxylated Substrates 110 2.3.3.1.2 Preparation of SAM 110 2.3.3.2 Preparation of Other Additives Used in the Enzyme Assay 112 2.3.3.2.1 Preparation of 1 M KCl in 2 % HCl 112 2.3.3.2.2 Preparation of Méthylation Products of the Enzyme Reaction 112 2.3.3.2.3 Preparation of Divalent Cations 113 2.3.3.2.4 Preparation of Inhibitors 113 2.3.3.3 Enzyme Assays 113 2.3.3.4 Calculations of Enzyme Activity in Pkat 114 2.3.3.5 Product Identification 115 2.3.3.Ô Protein Determination 116 2.3.3.6.1 Preparation of Protein Reagents (Bradford’s Reagent) 116 2.3.3.6.2 Protein Assay 116 2.3.4 Purification of Methyltransferases from A. altissima Suspension Cultures 117 2.3.4.1 Removal of Aromatic Compounds with XAD-2 resin 117 2.3.4.2 Ammonium Sulphate [(NH 4)2S04] Precipitation of Proteins 117 2.3.5 Further Purification of the 40 % - 70 % (NH 4)2S04 Protein Precipitate 118 2.3.5.1 Method 1: Desalting the 40 % - 70 % (NH4)2S04 Protein Precipitate 118 2.3.5.2 Method 2: Diethylaminoethyl (DEAE) Cellulose Anion Exchange Chromatography 118 2.3.5.3 Method 3: Hydroxylapatite Column Chromatography 119 2.3.5.4 Method 4: Sephadex G-50 Gel Filtration Chromatography 120 2.3.5.5 Method 5: Sephacryl S-200 Gel Filtration Chromatography 120 2.3.5.6 Method 6: Mono-Q Anion Exchange Chromatography 120 2.3.5.7 Method 7: Q-Sepharose Fast Flow Anion Exchange Chromatography 121 2.3.5.8 Method 8 : Gel Filtration on Pharmacia Superose 12 HR 10/30 Column 121 2.3.6 Characterisation of the 1-HMT Enzyme 122 2.3.6.1 Molecular Weight Determination 122 2.3.6.1.1 Molecular Weight Determination by the Gel Filtration Method 122 2.3.6.1.2 Molecular Weight Determination by Sodium Dodecylsulphate Poly acrylamide Gel Electrophoresis 123 2.4 Identification and Characterisation of Reference Compounds, Enzymes Substrates and Products Employed in the Study of A. altissima Cell Suspension Cultures. 126 2.4.1 Chromatographic Techniques 126 2.4.1.1 TLC of Canthin- 6-one Alkaloids 126 2.4.1.2 TLC of Coumarins 127 2.4.1.3 HPLC of Canthin- 6-one Alkaloids 128 2.4.1.4 HPLC of Coumarins 128 2.4.2 Spectroscopic Techniques 129 2.4.2.1 Fluorescent Spectra of l-hydroxycanthin- 6-one 129 2.4.2.2 UV, MS and NMR Spectra of Canthin- 6-one Alkaloid Standards Used as Reference Compounds, Enzyme Substrates and Products 129 2.4.2.3 UV, MS and NMR Spectra of Coumarin Standards Used as Reference Compounds, Enzyme Substrates and Products 131 3. RESULTS 133 3.1 Development of an Enzyme Assay for A. altissima Methyltransferase Enzymes and the Characterisation of Products of the Méthylation Reaction 134 3.1.1 Development of Enzyme Assay 134 3.1.2 Recovery of Methylated Products 134 3.1.2.1 Determination of levels of 1-Methoxy- canthin- 6-one Recovered During Product Extraction 135 3.1.2.2 Determination of the Reproducibility of the Product Extraction Procedure 136 3.1.3 Determination of Background Radioactivity 137 3.1.4 Determination of the Reproducibility of the Assay Procedure 138 3.1.5 The Sulphydryl Group Requirement 140 3.1.6 Product Identification 142 3.1.6.1 Identification of l-Methoxycanthin- 6-one 142 3.1.6.2 Identification of Methylated Coumarins 144 3.1.6.2.1 Identification of the Products of Aesculetin Méthylation 145 3.1.6.2.2 Identification of the Products

10 of Fraxetin Méthylation 147 3.2 Time Course Studies of A. altissima Cell Suspension Cultures 150 3.2.1 Growth, Canthin- 6-one Alkaloid Production and Corresponding Canthin- 6-one Alkaloid Methyltransferase Activity in Cell Suspension Cultures of A. altissima 151 3.2.1.1 Cell Line 1 151 3.2.1.1.1 Growth of Cells 151 3.2.1.1.2 Alkaloid Production 151 3.2.1.1.3 1-HMT Activity 153 3.2.1.2 Cell Line 2 153 3.2.1.2.1 Growth of Cells 153 3.2.1.2.2 Alkaloid Production 153 3.2.1.2.3 1-HMT Activity 155 3.2.2 Production of Methylated Coumarins and Corresponding Methyltransferase Activity in Cell Suspension Cultures of A. altissima 155 3.2.2.1 Cell Line 1 156 3.2.2.1.1 Coumarin Production 156 3.2.2.1.2 CMT Activity 158 3.2.2.2 Cell Line 2 158 3.2.2.2.1 Coumarin Production 158 3.2.2.2.2 CMT Activity 160 3.2.2.2.3 Méthylation of Coumarins by the CMT Enzyme 160 3.3 An Appraisal of Potential Purification Procedures for the 1-HMT Enzyme in A. altissima Cell Suspension Cultures 162 3.3.1 Purification Sequence la 164 3.3.2 Purification Sequence 2a 170 3.3.3 Purification Sequence 3a 173

11 3.3.4 Purification Sequence 4a 174 3.3.5 Purification Sequence 5a 177 3.3.6 The Choice of a Perfunctory Purification Procedure 180 3.4 Characterisation of the 1-HMT Enzyme of A. altissima Cell Suspension Cultures 183 3.4.1 Optimum Conditions for Enzyme Activity 183 3.4.1.1 Optimum Protein Levels 184 3.4.1.2 Optimum Incubation Time 187 3.4.1.3 Optimum Temperature 187 3.4.1.4 Activation Energy 192 3.4.1.5 Optimum pH 195 3.4.2 Michealis-Menten Constants (K^) for Enzyme Substrates 198 3.4.2.1 for SAM 201 3.4.2.2 K„, for l-Hydroxycanthin- 6-one 204 3.4.3 Inhibitor Studies 207 3.4.3.1 The Effect of Divalent Cations on Enzyme Activity 208 3.4.3.2 The Effect of Enzyme Inhibitors on Methyltransferase Activity 210 3.4.3.3 The Effect of Reaction Products on Enzyme Activity 212 3.4.3.4 Determination of the Inhibition Constant (KJ for SAH 215 3.4.3.4.1 Determination of the Inhibition Constant (KJ for SAH Using the Dixon Plot 215 3.4.3.4.2 Determination of the Inhibition

Constant (Kj) for SA H Using the Lineweaver-Burk Plot 217 3.4.4 Determination of the Molecular Weight of the

12 1-HMT Enzyme 220 3.4.4.1 Molecular Weight Determination by Gel Filtration on a Pharmacia Superose 12 HR 10/30 Column 221 3.4.4.2 Molecular Weight Determination by SDS-PAGE 223 3.4.5 Stability Studies 225 3.4.6 Méthylation of Hydroxylated Canthin- 6-one Substrates by the Canthin- 6-one Methyltransferase Enzymes from various A. altissima Cell Lines 227 3.5 The CMT Enzyme of A. altissima Cell Suspension Cultures 229 3.5.1 Partial Purification of the CMT Enzyme 229 3.5.2 Méthylation of Hydroxylated Coumarin Substrates by the CMT Enzyme from Ailanthus altissima Cell Suspension Cultures 229 3.5.3 Méthylation of Coumarin Substrates by Methyltransferases from various A. altissima Cell Lines 233 4. DISCUSSION AND CONCLUSIONS 235 4.1 The l-Hydroxycanthin- 6-one Methyltransferase Enzyme (1-HMT) of Ailanthus altissima Cell Suspension Cultures 236 4.1.1 The Possible Application of 1-HMT in the General Méthylation of Compounds 236 4.1.2 The Methyl ating Ability of A. altissima Cell Suspension Cultures: The Méthylation of 1 -Hy droxycanthin- 6-one 238 4.1.2.1 A Comparison of the Canthin- 6-one Methylating Capacities of Cell Lines 1 and 2: A Summary of the Observations 238 4.1.2.2 The Divergence in Methylating Abilities of Cell Lines 1 and 2 of A. altissima: 1-Hydroxycanthin- 6-one Méthylation 240

13 4.1.3 Purification and Characterisation of 1-HMT 246 4.1.3.1 The Selection of the Methodology for Purification of 1-HMT 247 4.1.3.2 The Comparison of the Characteristics of 1-HMT with Other Methyltransferases 248 4.1.3.2.1 The Characteristics of 1-HMT 248 4.1.3.2.2 Optimum Temperature 249 4.1.3.2.3 Optimum pH 249 4.1.3.2.4 Kinetic Properties 250 4.1.3.2.5 Inhibitor Studies 251 4.2 The Coumarin Methyltransferase Enzymes of A. altissima Cell Suspension Cultures 254 4.2.1 A Comparison of the Coumarin Methylating Abilities of 2 Cell Lines of A. altissima (Cell Lines 1 and 2): A Summary of the Observations 254 4.2.2 The Divergence of Methylating Abilities of Cell Lines 1 and 2 of A. altissima: Coumarin Méthylation 255 4.2.3 Purification and Characterisation of CMT 257 4.2.4 The Proposed Pathway for the Biosynthesis of Coumarins in A. altissima 257 4.2.4.1 The Méthylation of Coumarin Substrates by the CMT Enzyme of A. altissima 258 4.2.4.2 The Biosynthesis of Isofraxidin in A. altissima 259 4.3 Conclusions 261 4.4 Future Work 264 4.4.1 The Potential Use of 1-HMT in the General Méthylation of Compounds 264 4.4.2 The Biosynthesis of Coumarins in A. altissima 266 REFERENCES 269 LIST OF PUBLICATIONS 292

14 LIST OF FIGURES

Figure 1-1 Ailanthus altissima Tree 26 Figure 1-2 Ailanthus altissima Callus Cultures 28 Figure 1-3 Ailanthus altissima Cell Suspension Cultures 28 Figure 1-4 A Schematic Representation of Secondary Metabolites Originating from the Shikimate/Chorismate Pathway 54 Figure 1-5 The Biosynthesis of Tryptophan 55 Figure 1-6 The Proposed Biosynthetic Pathway for the Production of Canthin- 6-one Alkaloids in A. altissima Cell Suspension cultures 57 Figure 1 -7 The Role of a-Ketoglutarate in Canthin- 6-one Alkaloid Biosynthesis in Cell Suspension Cultures of A. altissima 60 Figure 1-8 The Biosynthesis of L-Phenylalanine from Chorismate 63 Figure 1-9 The Biosynthesis of Coumarins Lacking Oxygenation at the 7-Position e.g. Coumarin 64 Figure 1-10 The Biosynthesis of 7-Oxygenated Coumarins e.g. Umbelliferone 66 Figure 1-11 The Formation of Daphnetin form Umbelliferone 67 Figure 1-12 The Biosynthesis of Scopoletin, Fraxetin, Isofraxidin and puberulin 69

Figure 3.1-1 The Investigation of the Requirement of A. altissima Methyltransferases for Sulphydryl Group Protection 141 Figure 3.1-2 Identification of the Product of 1-Hydroxy- C-6-one Méthylation (by the 1-HMT Enzyme), Using TLC Techniques. 143 Figure 3.1-3 Identification of the Products of Aesculetin Méthylation (by the CMT Enzyme), Using TLC Techniques. 146 Figure 3.1-4 Identification of the Products of Fraxetin Méthylation (by the CMT Enzyme), Using TLC Techniques. 148 Figure 3.2-1 Time Course Study of Alkaloid Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell Line 1) 152 Figure 3.2-2 Time Course Study of Alkaloid Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell Line 2) 154 Figure 3.2-3 Time Course Study of Coumarin Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell Line 1) 157 Figure 3.2-4 Time Course Study of Coumarin Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell Line 2) 159

15 Figure 3.3-1 Purification Sequences Used for the Extraction and Purification of the 1-HMT Enzyme from A. altissima Cell Suspension Cultures 163 Figure 3.3-2 The Elution Profile of the Partially purified A. altissima Methyltransferase Enzyme from the DEAE-Cellulose Column 166 Figure 3.3-3 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Mono-Q Column 167 Figure 3.3-4 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Q-Sepharose Column 168 Figure 3.3-5 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima from the Superose 12 HR Column 169 Figure 3.3-6 The Elution Profile of the Partially Purified A. altissima Methyltransferase Enzyme from the Hydroxylapatite Column 172 Figure 3.3-7 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures, from the Sephadex G-50 Column. 176 Figure 3.3-8 The Elution Profile of the Partially Purified A. altissima Methyltransferase Enzyme from the Sephacryl S-200 Column 179 Figure 3.4-1 The Effect of Protein Levels on 1-HMT Activity (1) 185 Figure 3.4-2 The Effect of Protein Levels on 1-HMT Activity (2) 186 Figure 3.4-3 The Effect of Incubation Time on 1-HMT Activity (1) 188 Figure 3.4-4 The Effect of Incubation Time on 1-HMT Activity (2) 189 Figure 3.4-5 The Effect of Temperature on 1-HMT Activity (1) 190 Figure 3.4-6 The Effect of Temperature on 1-HMT Activity (2) 191 Figure 3.4-7 The Arrhenius Plot for the 1-HMT Enzyme (1) 193 Figure 3.4-8 The Arrhenius Plot for the 1-HMT Enzyme (2) 194 Figure 3.4-9 The Effect of pH on 1-HMT Activity (1) 196 Figure 3.4-10 The Effect of pH on 1-HMT Activity (2) 197 Figure 3.4-11 A Lineweaver-Burk Double Reciprocal Plot for the Determination of the of SAM 202 Figure 3.4-12 The Effect of SAM Concentration on 1-HMT Activity 203 Figure 3.4-13 A Lineweaver-Burk Double Reciprocal Plot for the Determination of the of 1-Hydroxycanthin- 6-one 205 Figure 3.4-14 The Effect of 1 -Hydroxycanthin- 6-one Concentration on 1-HMT Activity 206 Figure 3.4-15 The Effect of Divalent Cations on 1-HMT Activity 209 Figure 3.4-16 The Effect of Inhibitors on 1-HMT Activity 211 Figure 3.4-17 The Méthylation of 1 -Hydroxycanthin- 6-one to 1 -Metoxycanthin- 6-one with SAM as Methyl Group Donor 213

16 Figure 3.4-18 The Effect of the Reaction Products (SAH and 1-Methoxycanthin- 6-one) on 1-HMT Activity 214 Figure 3.4-19 An Investigation of the Competitive Inhibition of SAM by SAH. The Dixon Plot was Used to Determine the Ki for SAH 216 Figure 3.4-20 An Investigation of the Competitive Inhibition of SAM by SAH. The Lineweaver-Burk Plot was used to Determine the K, for SAH 219 Figure 3.4-21 The Elution Volume of Protein Standards from the Pharmacia Superose 12 HR 10/30 Column, Using Running Buffer B(l). 222 Figure 3.4-22 The Migratory Profile of the Purified 1-HMT Enzyme of A. altissima during SDS-PAGE 224 Figure 3.4-23 Stability Studies of 1-HMT 226 Figure 3.5-1 The Méthylation of Coumarin Substrates 231

Figure 4-1 The Biosynthesis of Isofraxidin from Aesculetin in Cell Suspension Cultures of A. altissima 260

17 LIST OF TABLES

Table 1-10 Canthin- 6-one Alkaloids Produced by A. altissima 31 Table 1-11 Canthin-6-one-3-N-Oxides Produced by A. altissima 31 Table 1-12 p-Carboline Alkaloids Produced by A. altissima 32 Table 1-13 Quassinoids Produced by A. altissima Trees 33 Table 1-14 Properties of O-Methyltransferases of Plant Origin 82 Table 1-15 Properties of N-Methyltransferases of Plant Origin 92 Table 2-10 Constituents of Incubation Mixes Used for Routine Assays in a Total Volume of 130 pi 109 Table 2-11 Constituents of Incubation Mixes Used for Standard Assays in a Total Volume of 130 pi 109 Table 2-12 Rf Values of Canthin- 6-one Alkaloid Standards 127 Table 2-13 Rf Values of Coumarin Standards 127 Table 2-14 Retention Times of Canthin- 6-one Alkaloid Standards 128 Table 2-15 Retention Times of Coumarin Standards 128 Table 3.1-10 Determination of Levels of 1-Methoxycanthin- 6-one Recovered by Product Extraction During Enzyme Assays 135 Table 3.1-11 Determination of the Level of Background Radioactivity 138 Table 3.1-12 An Investigation of the Reproducibility of the Assay Procedure Used for the 1-HMT Enzyme 139 Table 3.3-10 Purification of the 1-HMT Enzyme of A. altissima Using Sequence la 170 Table 3.3-11 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 2a 173 Table 3.3-12 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 3a 174 Table 3.3-13 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 4a 177 Table 3.3-14 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 5a 180 Table 3.4-10 Méthylation of Canthin- 6-one Substrates by Canthin- 6-one Methyltransferase Enzymes from Various A. altissima Cell Lines 228 Table 3.5-10 Méthylation of Coumarin Substrates from Sequence 1, by the Partially Purified CMT Enzyme from A. altissima (Cell line 2) 232 Table 3.5-11 Méthylation of Coumarin Substrates from Sequence 2, by the Partially Purified CMT Enzyme from A. altissima (Cell Line 2) 232 Table 3.5-12 Méthylation of Coumarin Substrates by CMT Enzymes from Various A. altissima Cell Lines 234

18 LIST OF ABBREVIATIONS

AMP Adenosine monophosphate AMT Aesculetin methyltransferase a Alpha BSA Bovine serum albumin P Beta P-MCE Beta-mercaptoethanol C-6-One Canthin- 6-one C Carbon Ci Curie cm Centimetre p-CMB para-Chloromercuric benzoate CMT Coumarin methyltransferase enzyme CNS Central nervous system Co Company CoA Coenzyme A Cu^+ Copper ion CUCI2 Copper chloride [] Concentration of cold Not radiolabelled °C Degrees celsius 2,4-D 2,4-Dichlorophenoxyacetic acid DEAE Diethylaminoethyl dpm Disintegrations per minute DTT Dithioerythritol dW Dry weight Ô delta E Enzyme e.g. For example ES Enzyme-Substrate complex et al And others FMT Fraxetin methyltransferase FPLC Fast protein liquid chromatography g gram HCl Hydrochloric acid 1-HMT SAM: l-Hydroxycanthin- 6-one Methyltransferase hot radiolabelled HPLC High pressure liquid chromatography HR High resolution H2SO4 Sulphuric acid I Inhibitor lA lodoacetamide lAA Indole acetic acid IBA Indole butyric acid

19 i.e. That is 9KB Carcinoma cells of the nasopharynx Ki Inhibitor constant Kg Kilogram KJ Kilojoule 1 Litre L Laevorotatory m Milli M Mole or Molar mA Milliampere MgCI, Magnesium chloride MgS04 Magnesium sulphate min. minute ml millilitre mg milligram Mg'+ Magnesium ion Mhz Megahertz mM Millimolar Mn^+ Manganese ion MnCl 2 Manganese chloride MS Mass spectroscopy MS Murashige and Skoog M.Wt Molecular weight NaCl Sodium Chloride NaH^PO^ Sodium dihydrogen phosphate Na^HPO^ Di sodium hydrogen phosphate Na^PO^ Sodium phosphate N Nitrogen NEM N-ethylmaleimide (NH4)2S04 Ammonium sulphate nm Nanometre nmr Nuclear magnetic resonance N-MT N-methyltransferase 0 Ortho O Oxygen OH Hydroxy O-MT 0-Methyltransferase KCl Potassium chloride KCN Potassium cyanide / Prime -1 Per P Para P388 Lymphocytic leukaemia from mice PAL Phenylalanine ammonia lyase Pkat Picokatal

20 PNA Polyneuridine aldehyde % Percentage rpm revolutions per minute S Sulphur or Substrate (S)- (S)-enantiomer SAH S-Adenosylhomocysteine SAM S-Adenosylmethionine SD Standard deviation SDS-PAGE Sodium dodecylsulphate polyacrylamide gel electrophoresis SH Thiol TCA Tricarboxylic acid TEMBD Tetramethylene diamine TLC Thin layer chromatography Tris-HCl Tris hydroxymethylaminomethane-HCl Mg microgram Ml microlitre pm micrometre pM micromolar UV Ultraviolet V ^ m ax Maximum velocity v/v Volume by volume w/v Weight by volume Y gamma

21 ABSTRACT

Ailanthus altissima cell suspension cultures produce significant levels of the biologically active canthin- 6-one alkaloids as well as the coumarins scopoletin and isofraxidin . Detailed time course studies of two cell lines of A. altissima were carried out in order to determine the growth, alkaloid production patterns and l-hydroxycanthin- 6- one methyltransferase (1-HMT) activities. These studies showed that cell line 1 produced 1-methoxycanthin- 6-one as the major constituent with canthin- 6-one and 1- hydroxycanthin- 6-one as minor constituents, whilst cell line 2 produced canthin- 6-one as the major constituent with 1-methoxycanthin- 6-one and 1 -hydroxycanthin- 6-one as minor constituents. The factors which could possibly regulate production of 1- methoxycanthin- 6-one in cell cultures of A. altissima were discussed. 1-HMT was extracted from cell suspension cultures of A. altissima and purified to a single protein using standard techniques. The enzyme was characterised as a monomer of molecular weight 60,000 - 63,000 Dalton. The optimum conditions for enzyme activity were determined and optimum substrate concentrations were obtained from the Michealis-Menten constants for SAM and 1-hydroxycanthin- 6-one. Inhibitor studies revealed that 1-HMT was inhibited by the products of the méthylation reaction, 1-methoxycanthin- 6-one and S-adenosylhomocysteine (SAH). 1-HMT was also inhibited by potassium cyanide and SH group inhibitors, iodoacetamide, N- ethylmaleimide and p-chloromercuric benzoate. The ability of the partially purified enzyme from various A. altissima cell lines to methyl ate 1 and lO-hydroxycanthin- 6-one was also investigated. These studies indicate that A. altissima cell lines vary in their specificity to the two hydroxylated canthin- 6-ones. The coumarin methyltransferase enzyme (CMT) from A. altissima cell suspension cultures was isolated and separated from the 1-HMT enzyme. Time course studies of the production of the identifiable methylated coumarins (scopoletin and isofraxidin ) as well as the corresponding methyltransferase activities were also carried out.

22 A Preliminary investigation of the ability of CMT to methylate hydroxylated coumarins was also done. CMT was found to monomethylate aesculetin to yield scopoletin but not isoscopoletin. This enzyme also had the ability to further methylate isoscopoletin (not found in A. altissima cell cultures) to yield scoparone, but lacked the ability to further methylate scopoletin. CMT also monomethylated fraxetin to yield isofraxidin . Whilst this enzyme had the ability to further methylate fraxidin (not found in A. altissima cell cultures) to yield 6,7,8-trimethoxycoumarin, it did not further methylate isofraxidin . These results indicate that a pathway for the production of isofraxidin from aesculetin, via scopoletin and fraxetin, exists in A. altissima cell cultures.

23 1. INTRODUCTION

24 1.1 Studies of Ailanthus altissima Ailanthus altissima Mill. Swingle has been used in this study due to its availability and the ease with which cell cultures may be developed. In addition to this, production of the canthin- 6-one alkaloids, is a major pathway occurring in cell cultures of A. altissima, which consequently produce these indole alkaloids in high yields. Cell cultures of A. altissima are therefore an ideal system for studying individual steps in the biosynthetic pathway for canthin- 6-one alkaloid production, particularly at the enzymatic level.

1.1.1 Ailanthus altissima as a Plant A. altissima belongs to the family Simaroubaceae, which as defined by Engler’s syllabus (Fo et al., 1992), consists of six sub-families with 32 genera and over 170 arboreous or shrubby species (Melchior, 1964). Most of the pharmacologically active species in the family Simaroubaceae are present in the sub-family Simarouboideae which consists of 22 genera. This subfamily is known to contain p-carboline and canthin- 6-one alkaloids as well as quassinoids (Simao et al., 1991; Fo et al., 1992).

1.1.1.1 A. altissima Tree A. altissima Mill. Swingle, Synonyms: A. glandulosa Desf., A. giraldii Dode and the tree of heaven, originated in China, but has been introduced into many countries including India, Japan and Australia (Anderson et al., 1983). A. altissima has also been established as an ornamental tree in cities throughout Europe and North America, where it has proved to be an excellent tree for planting in towns due to its display of pinnate leaves, colourful fruits and particularly for its apparent tolerance to city pollution (Swingle, 1916). A. altissima, shown in Figure 1-1, is a large deciduous, dioecious tree, frequently 50 to 70 feet high with a trunk of 2 to 3 feet in diameter. It has a rounded head of branches and many grey fissures mark the older bark. The I eaves are pinnate, 1 to 1.5 feet long and consist of 15 to 30 leaflets; leaflets are 3 to 6 inches long, ovate with the margin almost entire. The leaves of the male plant have a

25 FIGURE 1-1 Ailanthus altissima Tree

26 characteristic foetid odour. Flowers are in terminal panicles and the fruit consists of 3 to 5 keys, which form attractive orange to red clusters in August and September (Roberts, 1991). A. altissima is usually cultivated either from ripe seeds or from suckers that thrive in any good soil. Since it is preferable to produce female plants that do not have the undesirable foetid odour, propagation from root cuttings is the more popular method (Roberts, 1991).

1.1.1.2 A. altissima Cultures Germinated seeds of A. altissima readily form callus and cell suspension cultures when treated with the appropriate growth regulators. Callus and cell suspension cultures of A. altissima are shown in Figures 1-2 and 1-3 respectively. When examined microscopically, these cultures were found to consist of undifferentiated cells, either single or in small aggregates of 2 to 8 cells. Many of the aggregates showed incomplete cell division typical of several in vitro cultures. Staining the cells with Dragendorff’s reagent demonstrated the presence of both alkaloid accumulating and non-accumulating cells. Alkaloid production by cell suspension cultures was in the range of 0.55 to 1 mg . g ' fresh weight of cells. The alkaloids remained essentially within the cells with only trace amounts present in the medium (Anderson et ah, 1983).

1.1.2 The uses of A. altissima in Traditional Medicine Ailanthus species have been used historically in traditional Chinese medicine. A. exelsa was used specifically for the treatment of respiratory problems (Mehta and Patel, 1959), whilst A. malabarica was more widely used in the treatment of dyspepsia, bronchitis, ophthalmic diseases and snake bite (Khan, et a l, 1982). Throughout the Far East, various parts of A. altissima were considered to be of medicinal value. The fruit, the root or the stem have been used in the treatment of dysentery or leucorrhea (Steck, 1972). A. altissima has been used in traditional medicine for the treatment of enteric

27 FIGURE 1-2 Ailanthus altissima Callus Cultures

FIGURE 1-3 Ailanthus altissima Cell Suspension Cultures

jf y

28 infections of various origins (Document of American Herbal Pharmacology, 1975). A. altissima is also noted for anthelmintic, insecticidal and antibacterial properties (Ohmoto et al., 1976; Varga et al., 1980, 1981; Roberts, 1991). Like A. malabarica, A. altissima has also been used for the treatment of dyspepsia, bronchitis, ophthalmic diseases, snake bite and as a taeniafuge (Chopra et al., 1956; Khan et al., 1982). Trees harvested for medicinal purposes were usually felled in the spring or autumn and the bark removed and dried in the sun. Extracts of the bark have been used in the treatment of anaemia and as a taeniafuge (Roberts, 1991).

1.1.3 Secondary Metabolites from the Intact Plant ofA. altissima The following classes of secondary metabolites have been reported as occurring in the wood, root bark and bark of A. altissima: alkaloids, quassinoids, and quinones (Anderson et al., 1983).

1.1.3.1 Alkaloids Two categories of indole alkaloids were identified in A. altissima wood and root bark, i.e. the non-iridoid p-carbolines (Table 1-12) and the canthin- 6-one alkaloids (Tables 1-10 and 1-11). Canthin- 6-one was the major constituent of the root bark and wood (Anderson et al, 1983).

1.1.3.2 Quassinoids Quassinoids represent a class of degraded tetracyclic triterpenoids commonly found in Simaroubaceae species (Jaziri et al., 1988). Quassinoids are the major constituents of the bark of A. altissima trees. To date more than 20 quassinoids (Table 1-13) have been isolated from various parts of A. altissima and ailanthone is the major quassinoid constituent (Tang and Eisenbrand, 1992).

1.1.3.3 Quinones The quinones isolated from the whole plant of A. altissima include 2,6- dimethoxybenzoquinone (Souleles and Kokkalou, 1989) and [3,3-dimethylallyl]-quinol-

29 2-one (Anderson et al., 1983).

1.1.4 Secondary Metabolites from A. altissima Cell Cultures A. altissima cultures have a remarkable ability to produce high yields of canthin-6-one alkaloids, i.e. 100 fold greater than the whole plant in which they are normally found as very minor constituents (Anderson et al., 1987 a). In contrast to their high alkaloid producing ability these cultures produce quassinoids very poorly (Jaziri, 1987). A. altissima callus and suspension cultures produced high yields of canthin-6- one alkaloids at levels of 1.38 % and 1.27 % of the dry weight of cells respectively. These yields are consistently higher than those values reported in literature for alkaloid yields from the whole plant. The root bark was reported to contain 0.01 % of total alkaloid and the wood 0.001 % of total alkaloid (Anderson et a/., 1983). Other secondary metabolites found in cell cultures of A. altissima include quassinoids and coumarins.

1.1.4.1 Alkaloid Production Three groups have been involved in the recent studies of A. altissima in tissue culture. Anderson et al. (1983), developed callus cultures from sterile germinated seedlings. Callus induced from explants of the hypocotyl and cultures were maintained in Murashige and Skoog (M & S) basal medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D) (1 mg . 1'), kinetin (0.1 mg . r ‘) and sucrose (5 %), at 25 °C and under continuous illumination. Suspension cultures were maintained under the same conditions, with continuous agitation (120 rpm). The cells were subcultured every 28 to 30 days. Alkaloid yields of 100 mg . 1 ' were obtained; of which 62.5 mg . 1 ' was 1 -methoxycanthin-6-one, 20 mg . 1* was canthin-6-one and the rest was 1 -hydroxycanthin-6-one. This was the first report of canthin-6-one alkaloids from plant cell cultures of species in the family Simaroubaceae.

30 TABLE 1-10 Canthin-6-one Alkaloids Produced by A. altissima

Alkaloid R. R: R. R 5 Source R ef

Canthin-6 -one HHH H * * 1,2,3

l-Methoxy-C-6 -one OCH3 H H H * A 1,3,4

1 -Hydroxy-C-6 -one OH H H H * * 3,4,5

5-Hydroxy-C-6-one HHH OH * 3

2-Hydroxy-C-6-one HOH H H * 3

5-Methoxy-C-6-one H HH OCH3 A 2 ,

4-Hydroxy-C-6-one H H OH H * 3

5-Hydroxymethyl-C-6-one HHH CHnOH * 6

4,5-Dlhydrocanthin-6-one

TABLE 1-11 Canthin-6-one-3-N-Oxides Produced by A. altissima R

Alkaloid R Source R ef

Canthin-6-one-3N-Oxide H A * 2,3

1 -Methoxycanthin-6-one-3N-oxide OCH 3 * * 2,3

Legend: Tissue culture * ; Wliole plant * References (Ref): 1. Varga et al. (1980); 2. Ohmoto et al. (1981 a,b); 3. Crcspi-Perellino et al. (1986 a,b); 4. Souleles and Waigh (1984); 5. Khan and Shamsuddin (1981);6 . Ohmoto and Koike (1984).

31 TABLE 1-12 p-Carboline Alkaloids Produced by A. altissima

Alkaloid Source R, R: R3 R. Ref

P-Carboline-1- CjH^COOH HHH * $ 1,2,3 propionic acid 5

1 -( 1 -Hydroxy-2-methoxy) CH-CHjOH OCHj H H $ 1 ,2 . ethyl-4- 3 methoxy-p-carboline OCHj

1 -(2'-Hydroxyethyl)-4- C^H^OH OCHj H H* 1 ,2 . methoxy-p-carboline 3

4-methoxy-l-vinyl-P- CHCHj OCHj HH * 7 carboline

1 -(Carboxymethyl)-4- COOCHj OCH j H H * ♦ 1,3, methoxy-P-carboline 5

l-(Acetyl)-4-methoxy-p- COCHj OCH j H H * 3 carboline

1-(Methoxycarbonyl)-4,8- COOCH j OCH j H OCH j $ 6 dimethoxy-P-carboline

1 -(Melhoxycarbony 1)-P- COOCH j HH H * 6 Carboline

1-(r,2'-dihydroxyethy l)-4- CH-CH2 OH OCH j H H * 2,3 methoxy- P-carboline OH

6 -Methoxy-P-carboline-1 - COOCH j H OCH j H 4 carboxylic acid methyl ester

1 -Carbamoyl- P-Carboline CONH, H H H * 4

1 -Carbomethoxy-P-carboline COOCH j H HH $ 4,6

Legend: Tissue culture * ; Whole plant *

References(Ref): 1. Vargaet al. (1980); 2. Vargaet al. (1981); 3. Ohmoto et al. (1981 a,b); 4. Ohmoto and Koike

(1984); 5. Crespi-Perellino et al. (1986 a,b); 6 . Souleles and Kokkalou (1989); 7. Souleles and Waigh (1984),

32 TABLE 1-13 Quassinoids Produced by A. altissima Trees

Quassinoid Structure (Reference)

R Amarolide H HO. Amaroiide acetate Ac (Casinovi et ai, 1965;

Stôcklin et al., 1970) Me” H

Ailanthone (Casinovi et al., 1964; Naora et ai, 1982)

Glaucarubinone Me I (Gaudemer and Polonsky, 1965) O3C- C - Et I OH

13(21)-DehydrogIaucarubinone

(Tang and Eisenbrand, 1992) Ma O . C - C - E t OH

33 Quassinoid Structure (Reference)

13(21 )-Dehydroglaucarubolone o9" CH; HO (Tang and Eisenbrand, 1992) Me OH

Chaparroiide OH (Mitchell et al., 1971) HO Me Ma

Chaparrinone o9" (Polonsky and Fourrey, 1964) o

Shinjulactone A o9" HO, CHj (Naora et a i, 1983) HO Me

Shinjulactone B HO (Furano et al., 1981) Me O

34 Quassinoid Structure (Reference)

HO Ma HO Shinjulactone C HO HO Me OH (Ishibashi et al., 1983 b) O. Me Ma Me Me

HO Shinjulactone D HO .Ma HO (Ishibashi et al., 1983 a) HO

HO Shinjulactone E (Ishibashi et al., 1983 a) HO O

Shinjulactone F

(Ishibashi et al., 1984 a,b) O, ^ -We HO / W( — OH

Shinjulactone G (Ishibashi et al., 1984 c)

35 Quassinoid Structure (Reference)

Shinjulactone H HO ,Me (Ishibashi et a l, 1984 c) Me 1 Ma

Shinjulactone 1 HO

(Ishibashi et a i, 1984 b) Ma 1 Me

Shinjulactone J HO (Ishibashi et a i, 1984 b) HO Me I Me

AcO Shinjulactone K HO (Ishibashi et ai, 1984 b) Ma [ Ma

Shinjulactone L (Ishibashi et ai, 1985) OAc .Ma Ma I Ma HO,.

36 Quassinoid Structure (Reference)

Shinjulactone M \V' ° 'CHaOH " (Nimi et al., 1986)

HO Shinjulactone N CHz (Nimi et al., 1986) OH

Shinjudilactone ,Me (Ishibashi et a l, 1983 b)

Shinjuglycoside A HO Chaparrin-2-O-p-glucopyranoside (Yoshimura et a i, 1984) H0CH2

'oh \ Me HO OH

37 Crespi-Perellino et al. (1986 a,b) obtained callus cultures from sterile leaves, stem and shoots of A. altissima, using M & S medium with 2,4-D (1 mg . l '), at 28 °C and under continuous illumination. Suspension cultures were developed under the same conditions but were stirred continuously at 100 rpm at 28 °C in the dark. Cultures harvested at 25 days produced an average of 400 mg . 1* of alkaloid of which 90 % was canthin-6-one. The second most abundant alkaloid was 1-methoxycanthin- 6-one. This Italian group reported the isolation of eight canthin-6-one alkaloids (Table 1-10), namely: canthin-6-one, l-methoxycanthin-6-one, 5-hydroxycanthin-6-one, 1- hydroxycanthin-6-one, 2-hydroxycanthin-6-one, 4-hydroxycanthin-6-one, 4,5- dihydrocanthin-6-one and l-(carboxymethyl)-4-methoxy-p-carboline. Three of these alkaloids had not been previously found in nature, these include: 2-hydroxycanthin-6- one, 4-hydroxycanthin-6-one and 4,5-dihydrocanthin-6-one. These novel alkaloids represented 0.3 % of the total alkaloids and their production was of the order of 2 mg . ml ’. Aragozzini et al. (1988), prepared callus cultures from stems of A. altissima, using Gamborg’s agar medium with naphthalene acetic acid (4 mg . 1') and 2,4-D (0.2 mg . 1’) as phytohormones. Suspension cultures were prepared from callus cultures using a similar medium, but these cultures were continuously agitated at 120 rpm at 26 °C. These cultures yielded, on the average, 50 mg .1 ' of canthin-6-one, after thirty days of growth.

1.1.4.2 Quassinoid Production Jaziri et al. (1988) cultivated callus cultures from sterile stem and anther explants. Callus cultures were initiated and subsequently maintained on M & S medium supplemented with 0.1 pM kinetin, polyvinyl pyrollidone (1 %), charcoal (0.3 %) and an auxin: [Different types of auxins were tested, to select the most suitable. Auxins tested include: 1 pM 2,4-D, 20 pM indole acetic acid (lAA) or 20 pM indole butyric acid (IBA)]. The cultures were maintained at 24 °C under continuous illumination (2000 lux). Jaziri et al. (1988), reported that after the fifth transfer in medium containing 2,4-D, callus from stem explants produced ailanthone, when

38 transferred into media containing lAA or IB A. Anther explants, on the other hand, produced ailanthone only when maintained in medium containing 2,4-D; however in both cases ailanthone yields were generally low and averaged only about 15 pg . 100 g * fresh weight of callus culture on the average.

1.1.4.3 Coumarin Production Callus cultures of A. altissima are reported to produce the coumarins scopoletin and isofraxidin (Hay, 1987 and Roberts, 1991).

1.1.5 Studies on the Biological Activities of the Quassinoids and Canthin-6-one Alkaloids of A. altissima

1.1.5.1 Quassinoids Investigations into biologically active constituents have focused on the quassinoids since they exert marked biological activities (Polonsky, 1983). They reportedly exhibit antiprotozoal, anthelmintic, insecticidal, antileukaemic and cytotoxic activities. Quassinoids isolated from A. altissima have shown the following biological activities:

(1) Anti-amoebic Activity Extracts of the bark and fruit of A. altissima were traditionally used in the treatment of dysentery. This activity can be attributed to the major quassinoid constituent, ailanthone, found to exert potent anti-amoebic activity against Entamoeba histolytica both in vitro and in vivo (De Cameri and Casinovi, 1986). Other quassinoid constituents of A. altissima have also displayed anti-amoebic activity with the following IC50 values, expressed in (pg . ml ‘), ailanthone (0.04), ailanthinone (0.06) and glaucarubinone (0.025) as compared with a standard amoebicide, metronidazole (0.35) (Gilli n et a i, 1982; Wright et al., 1988).

39 (2) Anti-malarial Activity Crude extracts of A. altissima have also been found to be active against Plasmodium falciparum, in vitro and P. berghei in mice. The anti-malarial activity was attributed principally to the presence of the quassinoid ailanthone, which possesses an a-ketol group in ring A. Glaucarubinone, similar in structure to ailanthone, showed a similar activity. Ailanthone and glaucarubinone however were both highly toxic to mice at a dose of 9 mg . kg ’ . day '; their potential as future anti- malarials is therefore quite unlikely (O’Neill et al., 1986). However Suffness and Douros (1980), linked cytotoxicity to a free hydroxyl group at C 1, so drugs with low cytotoxicity may eventually be produced.

(3) Anti-cancer Activity Quassinoids are considered to be potential sources of new anti-cancer agents (Suffness and Douros, 1980). Ailanthinone, glaucarubinone and 13(21)- dehydroglaucarubinone displayed in vitro anti-cancer activity against the lymphocytic leukaemia system from mice (P388) and carcinoma cells of the nasopharynx (9KB) (Ogura, et al., 1977; Suffness and Douros, 1980). Investigation of the structure activity relationships of quassinoids suggests that the oxygen bridge at C-20 - C-11 or C-20 - C-13, and an ester function at C-15 are necessary for optimal activity in all of the above tests (Cassady and Suffness, 1980; Roberts, 1991).

1.2.5.2 Alkaloids Two categories of non iridoid indole alkaloids have been identified in A. altissima, i.e. the non-iridoid P-carbolines and the canthin-6-ones. These alkaloids are listed in Tables 1-10 to 1-12. They possess some interesting biological activities:

(1) Anti microbial activity Canthin-6-one alkaloids, especially canthin-6-one, possess antibacterial and antifungal activity. The antibacterial activity of Zanthoxylum elephantiasis has been

40 attributed to canthin-6-one, which showed significant activity against Staphylococcus aureus, Klebsiella pneumoniae and Mycobacterium smegmatis (Anderson et al., 1983; Mitscher et al., 1972). This wide range of activity is apparently specific to canthin-6- one. The other canthin-6-one constituents of Z. elephantiasis (5-methoxycanthin-6- one, 4-methylthiocanthin-6-one, 4,5-dimethoxycanthin-6-one and benz-(4,5)-canthin-6- one), were without meaningful activity.

(2) Anti-cancer activity There are conflicting reports on the activity of canthin-6-one alkaloids as anticancer agents. An original report by Cordell et al. (1978), suggested that canthin- 6-one, 1-methoxycanthin-6-one and 5-methoxycanthin-6-one are not significantly active in the cytotoxicity test (9 KB system i.e. carcinoma cells of the nasopharynx). Anderson et al. (1983), however, found that canthin-6-one, 5-methoxycanthin-6-one and canthin-6-one-3N-Oxide have marked cytotoxic activity against guinea pig ear kératinocytes (GPK). Another study revealed that 11-hydroxycanthin-6-one and 1,11- dihydroxycanthin-6-one demonstrated significant antileukaemic activity in vitro against 9KB cells (Fukamiya et al., 1986). Further studies showed that lO-methoxycanthin-6- one and lO-hydroxycanthin-6-one are also active in the 9 KB cytotoxicity test system (Arisawa et al., 1983). The structure activity relationships of a number of canthin-6- one alkaloids isolated by Fukamiya et al., 1986 were investigated. The results suggested that either hydroxylation or méthoxylation of canthin-6-one alkaloids at C- 11 or C-10 is required for potent cytotoxicity (Fukamiya et al., 1986, 1987). A number of canthin-6-one alkaloids were tested for antileukaemic activity against the P 388 system i.e. (lymphocytic leukaemia) and of these only 1,11- dimethoxycanthin-6-one was shown to be active. On this basis, Fukamiya et al. (1986), concluded that oxygenation at C-1 and C-11 of canthin-6-one contributed significantly to this activity.

41 (3) Cyclic Adenosine Monophosphate (AMP) Phosphodiesterase Inhibitory Activity P-Carboline and canthin-6-one alkaloids demonstrate strong inhibitory effects on cyclic adenosine monophosphate phosphodiesterase (Sung et aL, 1984). Structure activity relationships were studied for 31 derivatives of p-carboline, two dimeric derivatives of p-carboline and 12 derivatives of canthin-6-one. P-Carboline derivatives possessing a methoxycarbonyl group and canthin-6-one derivatives with a hydroxymethyl group generally exerted a strong inhibitory effect on cyclic-AMP phosphodiesterase. Apparently, the presence of an oxygen atom at C-5 or at both C-4 and C-5 is essential for the inhibition of cyclic AMP phosphodiesterase by the canthin-6-one alkaloids. Some compounds isolated from A. altissima were tested for cyclic-AMP phosphodiesterase inhibitory activity, but only two of them, viz.: 1-(1- hydroxy-2-methoxy)-ethyl-4-methoxy-p-carboline (IC^Q: 4.6 x 10^ M) and 5- hydroxymethylcanthin-6-one (IC^g: 4.8 x 10 ^ M), had inhibitory activity of the same magnitude as the commonly used standard, papaverine (IC^o: 3.0 x 10 ^ M).

(4) Central Nervous System (CNS) Depressant Activity Crude extracts of the root bark and cell cultures of A. altissima were evaluated for their activity on the central nervous system. Both the crude extracts showed a clear CNS depressive activity, in mice, when administered intraperitoneally at the dose of 50 mg . kg '. The active constituent is 4,5-dihydrocanthin-6-one. The crude extract from the plant demonstrated a lower CNS-depressive activity than the extract of cell cultures but was highly toxic, causing death of the mice within 24 hours of treatment. Crude extracts from cell cultures of A. altissima, on the other hand showed no macroscopic signs of toxicity (Crespi-Perellino et aL, 1988).

42 1.2 Plant Cell Cultures as Alternatives to Intact plants Plant cell culture can be defined as growing sterile plants, tissues or cells on artificial media in vitro i.e. separate from the mother plant (Stafford, 1991). Culture of plant cells or tissue on defined media under controlled conditions allows manipulation of many facets of plant biology with relative ease. Plant cell cultures are potential resources for the commercial production of secondary metabolites and drugs (Zenk, 1978; Fowler, 1987; Phillipson, 1990 a,b). They have found useful application in organ, tissue and cell regeneration. Plant cell cultures have also been used in the study of metabolic pathways and their regulation.

1.2.1 The Commercial Value of Plants in the Pharmaceutical Industry A striking attribute of plants is the wide range of secondary metabolites formed throughout the plant kingdom. Plants with medicinal properties are in high demand in the pharmaceutical industry (Balandrin et aL, 1985). 40 % of the drugs presently in use are natural products or derived from natural products (Samuelson, 1982) and 119 plant derived chemical compounds of known structure are used as pharmaceutical agents (Farnsworth, 1991). In 1989, the Japanese output of herb and crude medicines was 137.7 billion Yen, an increase of 17 % over the preceding year. In 1990, the total market size of anti-cancer drugs vinblastine and vincristine was 3.3 billion Yen, a 10 % increase over the previous year. Sales of drugs obtained from Digitalis, e.g. digoxin were 2.3 billion Yen, an increase of 8 % over the previous year. The value of codeine phosphate, a narcotic cough remedy was 2.4 billion yen, as at 1991. Spices, condiments and beverages are also in high demand and are quite highly priced. These products used in the food industry owe their aroma and flavour to pharmacologically active secondary metabolites (Publication Division: CMC Co. Ltd., 1991). Plants also serve as a source of other chemical products that relate to pharmacy. Examples include: narcotics (opium from Papaver somniferum), stimulants (caffeine from Coffea arabica), insecticides (pyrethroids from Chrysanthemum cinerariaefolium), cosmetics and aromatics (various essential oils in plants) (Bajaj et at., 1988).

43 In the United States and Europe, large amounts of money are spent on the importation of medicinal plants. In the U.S. the purified opium alkaloids, codeine and morphine, cost between $650 and $1250 per kg, phorbol esters have a retail value of approximately $2000 per kg (Balandrin et aL, 1985) and the anticancer Catharanthus alkaloids had a wholesale value of about $20,000 per gm as far back as 1983 (Curtin, 1983). In Western Europe, the import trade in medicinal plants during 1980 was estimated as US $117 million in four countries, i.e. the UK, Netherlands, France and West Germany (Farnsworth, 1984). Despite the expense of medicinal plants to the pharmaceutical industry, they will continue to be important sources of novel agents. Alkaloids are one of the most important groups of pharmacologically active principles found in plants and quite a number of them are used medicinally for a wide range of pharmacological effects. Pertinent examples include; morphine and codeine (analgesic), emetine (antiamoebic), quinine (antimalerial) quinidine and ajmaline (antiarrhthmic), vinblastine, vincristine, harringtonine, campothecin (anticancer), reserpine, rescinnamine, (hypotensive), cocaine (local anaesthetic) and tubocurarine, (muscle relaxant) (Balandrin et aL, 1985). Whilst synthetic routes to many alkaloids are well established, economically viable processes exist for only a few of the medicinal alkaloids in common use, e.g. caffeine and ephedrine (Hay et aL 1988). Consequently morphine and codeine are still isolated from the latex of unripe seed pods of Papaver somniferum and quinine and quinidine are harvested from the bark of Cinchona trees. A large portion of the plant kingdom has not been surveyed for biologically active compounds. On-going research is therefore aimed at searching for new drug templates and reducing the production cost of plant derived drugs. It is expected that biotechnological advances will greatly help to achieve these goals.

1.2.2 The Application of Plant Cell Cultures in Drug Production Even though most plants from which important pharmaceuticals are isolated are grown on large scale plantations (Chattergee, 1978), an alternative means of

44 production would probably be of interest to the pharmaceutical industry, for reasons of convenience and cost. Production of secondary metabolites by plant cell cultures is a potential alternative to the traditional methods of cultivation.

1.2.2.1 Advantages of Plant Cell Cultures in Secondary Metabolite Production Factors that apparently give plant cell culture techniques an advantage over traditional methods of cultivation include the following (Fowler, 1983):

(1) Elimination of risks of [crop failure. (2) Reduction of transportation costs. (3) Freedom from geographical influences. (4) Elimination of environmental factors such as climatic and seasonal variation. (5) Freedom from disease. (6) More consistent product yield. (7) Improvement of quality of products. (8) Elimination of political and bureaucratic interference. (9) Flexibility of location for production.

The question however is, whether plant cell culture can actually take over a significant role in the pharmaceutical industry now or in the future. Over the last decade, the potential of plant cell cultures as an alternative commercially viable means of producing secondary metabolites has been the focus of intense investigation. In spite of the initial optimism, few commercial successes have been achieved, the chief constraints being that cultured cells tend to produce inferior yields of the desired metabolites in comparison to the whole plant (Heinstein, 1985; Staba, 1985). Nevertheless more than 30 compounds are known to accumulate in cultures at levels higher than in the parent plant. These cultures include several alkaloid producing species such as A. altissima (canthin-6-one alkaloids) (Anderson

45 et aL, 1987 a), Catharanthus roseus (ajmalicine and serpentine) (Zenk et aL, 1977) and Coptis japonica (berberine) (Satô and Yamada, 1984), which illustrate the biosynthetic potential of plant cell cultures. However in order for a process to be a viable commercial reality, it is essential that it can be successfully scaled up. To achieve this, many factors have to be considered. In recent years a great deal of interest has been focused on areas such as the design of bioreactors, mixing and aerating cultures, and maintaining sterility within the bioreactors (Fowler, 1986).

1.2.2.2 The Commercial Production of Drugs and Secondary Metabolites by Plant Cell Cultures Some secondary metabolites have been successfully produced on a commercial scale by plant cell culture techniques, whilst others are presently being investigated, because their commercial production by plant cell cultures, appears promising.

(1) Production of shikonin Shikonin is used medicinally for its anti-inflammatory and antiseptic properties (Yamada and Fujita, 1983; Tabata and Fujita, 1985). Shikonin compounds found in the roots of L. erythrorhizon have been used in traditional dyeing. The plant has also been used as a constituent of a herbal medicine known as Shiun-Ko (Takahasi and Fujita, 1991). The naphthaquinone shikonin, has been produced on a commercial scale, from suspension cultures of Lithospermum erythrorhizon by Mitsui pharmaceuticals in Japan (Fujita et aL, 1981). Production of shikonin compounds by plant tissue culture was originally investigated because the cultivation of L. erythrorhizon is difficult and the wild plant is almost extinct in Japan (Takahasi and Fujita, 1991). Cultures of L. erythrorhizon are reported to produce 1.5 g . 1‘ shikonin derivatives which represents a yield 15 times greater than that of the parent plant (Tsukada and Tabata, 1984). However the plant cell culture process has not been approved for the production of shikonin as a medicinal agent, thus shikonin obtained from plant cell culture is mainly used as a colorant in cosmetics.

46 (2) Production of Ginseng The roots of Panax ginseng C.A. Meyer (Araliaceae), has been highly priced from ancient times as a tonic and a precious medicine, which is effective for the treatment of gastrointestinal disorders, diabetes and poor blood circulation; it has been widely used as an adjuvant to prevent disorders, rather than as a medicine to cure disease, especially in China and Korea. The production of ginseng roots is however limited due to the cold, semi-dry and shaded habitat requirement (Ushiyama, 1991). Ginseng has been produced on a commercial scale in Japan, from plant cell cultures of Panax ginseng. Furaya et al. (1983), reported success in obtaining cultured tissues of P. ginseng, which accumulated as much ginsenoside as did the cultivated roots. The authors therefore suggested the possibility of large scale cell cultures; by 1989, ginsenosides were being produced by cell cultures (Fujita et al., 1989).

(3) Production of berberine Berberine in the form of berberine chloride is used as a medicinal for intestinal disorders (Matsubara and Fujita, 1991). Berberine is an isoquinoline-type alkaloid which was originally produced in Japan by extraction from the roots of Coptis japonica and the cortex of Phellodendron amurense. Production of berberine from these sources was however, halted due to economic reasons. Subsequently Yamada’s group (Yamada and Sato, 1981; Sato and Yamada, 1984), working with C. japonica cell cultures and using cell line selection techniques, were able to develop cell lines of C. japonica whose berberine content was higher than the whole plant.

(4) Production of tripdiolide and triptolide Tripdiolide and triptolide are active against leukaemias and human carcinoma of the nasopharynx (Kutney, 1985). Tripdiolide and triptolide are diterpene triepoxides produced by Tripterygium wilfordii, a shrub found in Taiwan, China, Korea and Japan. Natural resources of this plant are limited and they yield low levels of tripdiolide and triptolide. In addition.

47 total synthesis was found too complicated to produce sufficient amounts of these compounds, and so the use of tissue culture was considered. Kutney et aL (1980) developed cell suspension cultures of T. wilfordii which produced 3 to 16 times the amount of tripdiolide and triptolide produced by the intact plant, thus offering a potential for commercial production by tissue culture (Takayama, 1991).

(5) Production of sanguinarine Sanguinarine, a benzophenanthridine alkaloid, has been used as an oral hygiene product in the United States. The fact that a toothpaste producer, Colgate, has shown interest in this product, suggests its potential commercial value (Park et at., 1990). Sanguinarine has been produced in high yields by elicited cell cultures of Papaver somniferum (Park et aL, 1990).

(6) Production of taxol Taxol, produced by Taxus hrevifolia, has been found to exhibit activity against ovarian cancer (Kingston et aL, 1990). Production of taxol as well as other antitumour compounds by tissue culture is a subject of active research and it is hoped that production of these compounds by tissue culture will become a routine procedure, through improved understanding of biotechnology (Takayama, 1991).

1.2.3 Plant Cell Cultures in Biotransformation Reactions Another area of commercial importance involving the utilisation of plant cell and tissue culture is that of biotransformation. It has been shown that plant cell cultures can perform special biotransformation reactions of exogenous organic compounds added to the culture medium (Reinhard and Alfermann, 1980). However the chances of using plant cell cultures for biotransformation or as enzyme sources is dependent upon the commercial importance of the observed reaction and the chances of being developed on an industrial scale. Biotransformation reactions utilising plant cell culture are economically feasible, only if the process is not in competition with one that can be carried out by microorganisms.

48 Pertinent examples of biotransformation reactions in plant cell culture include the formation of cardenolides in Digitalis lanata cell cultures and the formation of vinblastine and vincristine in Catharanthus roseus cell cultures.

1.2.3.1 Formation of Cardenolides in Digitalis lanata Cell Cultures Microbes have the ability to readily hydroxylate aglycones, but hydroxylation at the C-12 position of the glycosides does not occur or is incomplete, thus the use of plant cell cultures is preferable. Cultures of Digitalis lanata do not synthesise cardiac glycosides but they are capable of carrying out the 12p-hydroxylation which is responsible for the conversion of digitoxin to digoxin. Reinhard and Alfermann (1980), screened D. lanata cell cultures for hydroxylation activity; when P-methyl digitoxin was used as substrate, it was completely transformed to p-methyl digoxin. This process has been scaled-up successfully (Berlin, 1988) but the commercial application has yet to be reported.

1.2.3.2 Biosynthesis of Vinblastine and Vincristine in Plant Cell Cultures by B iotransfor mation The dimeric alkaloids vinblastine and vincristine, are products of the coupling of vindoline and catharanthine, two of the major alkaloids found in the leaves of C. roseus. Vinblastine and vincristine occur at low levels in the leaves and are among the most expensive drugs on the pharmaceutical market. Consequently a major research goal has been the development of vinblastine and vincristine production from C. roseus tissue culture. Tissue cultures have however failed to produce these dimeric alkaloids, because they lack the ability to synthesise vindoline, an obligatory precursor to vinblastine and vincristine (Carew and Krueger, 1977; Endo et aL, 1987). Biotransformations by plant cell suspension cultures are therefore of considerable interest and the ability of cell suspension cultures of C. roseus to form vinblastine from catharanthine and vindoline has been demonstrated. The enzyme responsible for the coupling of vindoline and catharanthine to form dimeric alkaloids vinblastine and vincristine has been identified and isolated from cell suspension cultures of C. roseus

49 (Hamada and Nakazawa, 1991). A substantial yield of 3',4'-anhydrovinblastine, an immediate precursor of vinblastine has also been obtained from these cultures. Biotransformation of vinblastine to the more valuable vincristine has also been reported to occur in cell suspension cultures of C. roseus. Incubation of vinblastine in cell suspension cultures of C. roseus resulted in its conversion to vincristine which was the sole product after a period of two days (Hamada and Nakazawa 1991).

1.2.4 Plant Ceil Cultures in the Study of Biosynthetic Pathways Studies with cell suspension cultures revealed for the first time substantial insight into the biosynthetic pathways of different groups of natural products especially alkaloids. A few pertinent examples include the isoquinoline alkaloids (Herbert, 1992), and the indole alkaloids (Kutney, 1990). In spite of the progress made in plant cell culture through the use of production media, elicitors, as well as screening and selection techniques, it is not yet possible to manipulate the expression of desired secondary pathways. So long as this is the case, industrial interest will remain comparatively limited. A complete knowledge of the enzymology and molecular biology of secondary pathways could lead to the exploitation of the biochemical potential of plant cell cultures. Therefore present research in this field has been directed to unravelling factors which regulate important biosynthetic pathways, identifying the enzymes and cloning genes involved in these pathways (Berlin, 1988; Kutchan, 1993). Plant cell cultures possess certain advantages over differentiated plants for biosynthetic studies. These include the fact that cultures can be grown under standard conditions for short growth cycles and are not subject to seasonal variations. This ensures an all year round availability of biomass at the required stage of growth for enzyme extraction. In addition to this, plant cell cultures are less complex in organisation than the entire plant and hence permeability, translocation and segregation of precursors and products do not present the problems which are sometimes encountered in the whole plant (Anderson et al., 1985).

50 In the past, plant cell cultures have been used as efficient sources for the isolation of novel enzymes (Stockigt, 1979; Treimer and Zenk, 1979 a,b; Khouri et aL, 1988 b; Frenzel and Zenk, 1990 a,b; Rueffer et aL, 1990). Cultivated plant cultures can therefore be regarded as a valuable tool for the investigation of the biosynthesis of secondary metabolites. Cell suspension cultures are preferable to callus cultures for use in biosynthetic studies, due to the ease of administration of precursors and product extraction (Stockigt and Schiibel, 1988). Cultured cells, however, sometimes produce inferior yields of the desired metabolites in comparison to the whole plant and may not produce the same secondary metabolites as the parent plant. A typical example is the production of commercially important indole alkaloids from cell suspension cultures of C. roseus. The production of catharanthine and vindoline; precursors of the commercially important antineoplastic drugs, vinblastine and vincristine, has been investigated by several groups (Kutney et aL, 1976; Kurz and Constabel, 1985; Zenk and Deus, 1982; De Luca et aL, 1986). These investigations have established the fact that cell suspension cultures of C. roseus like the whole plant, produce catharanthin (Stockigt and Soil, 1980; Kutney et aL, 1980), but the production of vindoline the major plant alkaloid has never been unequivocally demonstrated. Several enzymes involved in vindoline biosynthesis have been isolated and characterised from etiolated seedlings (Balsevich et aL, 1986; De Luca et aL, 1987), leaves and shoot tips of C. roseus (Fahn et aL, 1985; De Luca et aL, 1987; De Carolis et aL, 1990; Fahn and Stockigt, 1990; Power et aL, 1990). It has been suggested that the genes for vindoline biosynthesis are merely repressed in callus cultures and not lost (Power et aL, 1990). Constabel et aL (1982), reported that, the ability to synthesise vindoline was restored in shoots regenerated from C. roseus Little Delicata callus. In spite of these inconsistencies which sometimes occur between secondary metabolite production in plant cell cultures and the whole plant, the use of cell suspension cultures have made possible the isolation and study of enzymes involved in pathways leading to the production of some pharmacologically useful natural products. This knowledge provides further insight into those factors which regulate

51 production in vitro, thereby furnishing the information necessary for genetic manipulation and the production of increased levels of secondary metabolites. A typical example of this is ajmaline biosynthesis. The biosynthesis of the monoterpenoid indole alkaloid ajmaline (an antiarrythmic drug) was a matter of speculative discussion for more than 20 years (Pfitzner and Stockigt, 1983). The establishment of cell suspension cultures of Rauwolfia serpentina Benth., capable of synthesising these alkaloids, made a detailed investigation regarding enzymatic formation possible (Stockigt et aL, 1981). The enzymes catalysing reactions in the main biosynthetic route to ajmaline have since been isolated. 3 side reactions leading to the formation of sarpagine (Pfitzner et aL, 1984; Stockigt and Schiibel, 1988), raucaffrinoline (Stockigt and Schiibel, 1988) and raucaffricine (Schiibel et aL, 1986), were also identified. The first 2 side reactions i.e. formation of sarpagine and raucaffrinoline, did not have a significant influence on ajmaline production in R. serpentina cultures, as they were present in trace amounts. Formation of raucaffricine on the other hand, appeared to effectively diminish ajmaline yield, with the pathway to ajmaline ending preferentially at the stage of raucaffricine. Raucaffricine has been reported to accumulate in cell suspension cultures at amounts of up to 1.2 g . l ' medium whilst ajmaline was produced in yields of about 0.3 g . 1^ medium (Schiibel et aL, 1986). There is a possibility that by the development of experimental methods to inhibit raucaffricine biosynthesis, the real hindrance to ajmaline formation can be identified and corrected (Stockigt and Schiibel, 1988).

1.2.4.1 Plant Cell Cultures in the Study of the Biosynthesis of Canthin-6-one Alkaloids Plant cell cultures of A. altissima have been very useful in the study of the biosynthetic pathway for the production of canthin-6-one alkaloids. Canthin-6-ones are among the simplest indole alkaloids and are present in a large number of plants (Allen and Holmsted, 1980). The parent compound, canthin- 6-one, was first isolated from Pentaceras australis (Rutaceae) and was subsequently

52 obtained from a number of plants in the Simaroubaceae, which is botanically close to the Rutaceae (Cordell, 1981; Waterman, 1983). In spite of the comparatively simple structure of the canthin-6-one alkaloids, work done on their biosynthesis was minimal. Until recently, the information available on the biosynthesis of this group of alkaloids was inadequate. In 1973, Hegnauer suggested that canthin-6-one alkaloids could be derived from one of two routes: (1) From a p-carboline intermediate, to which a C-2 unit derived from either acetate or pyruvate is added, or (2) From tryptamine, to which a C-4 unit is incorporated.

Cell cultures of Ailanthus altissima, high yielding in canthin-6-one alkaloids, have been useful as a model system for biosynthetic studies and for investigating the factors which regulate production of these alkaloids. Several aspects of the biosynthesis of canthin-6-one alkaloids have been investigated using plant cell cultures of A. altissima. These include: (1) Tryptophan as a precursor of the indole fragment of canthin-6-one (Anderson et aL, 1986; Crespi-Perellino et aL, 1986 b). (2) The intermediacy of p-carboline-1-propionic acid and the involvement of acetate (Aragozzini et aL, 1988). (3) The role of methionine as the methyl group donor for l-methoxycanthin-6- one (Anderson et aL 1987 b).

1.2.4.1.1 The role of Tryptophan and Tryptamine in the Biosynthesis of Canthin- 6-one Alkaloids Crespi-Perellino et al. (1986 a,b), suggested that the precursor for the alkaloid canthin-6-one is tryptophan. The aromatic amino acid tryptophan as well as tyrosine and phenylalanine are precursors of various classes of secondary metabolites including alkaloids. These alkaloids are derived from the Shikimate / Chorismate pathway outlined in Figure 1-4

53 FIGURE 1-4 A Schematic Representation of Secondary Metabolites Originating from the Shikimate/Chorismate Pathway

Shikimate ~ r ~ Chorismate

~ T -

Isochorismate Prephenate Anthraniiate

Tryptophan Tyrosine Phenylalanine f Î 1 7 T Cinnamate Indole Indole Indole Isoquinoline Betalins acetic alkyl Alkaloids alkaloids acid amines 1r i I I i Flavonoids Substituted Coumarins Lignin coumarins precursors

Legend: IS = Isochorismate synthase CM = Chorismate mutase AS « Anthraniiate synthase (Poulsen and Verpoorte, 1991). Chorismate is a major intermediate in the Shikimate / Chorismate pathway. Chorismate is the precursor of the biosynthesis of the secondary metabolites, and serves as the substrate for a number of enzymes involved in the biosynthesis of aromatic compounds. These enzymes include: 1. Chorismate mutase which catalyses the conversion of chorismate to prephenate, the precursor of tyrosine and phenylalanine. 2. Anthraniiate synthase which is responsible for the conversion of chorismate to anthraniiate, the precursor of tryptophan. 3. Isochorismate synthase which is responsible for the conversion of chorismate to isochorismate, from which anthraquinones and vitamin Kj are produced.

Biosynthesis of Tryptophan FIGURE 1-5 The Biosynthesis of Tryptophan

COOH

NH ^ ^ cooH (p)-ocH; 0 ty-ocHja

OH OH OH OH Anthranilic 5 -Phosphori bosyI-1 - N-(5'-phosphoribosyl)- acid diphosphate anthranilic acid 1®

OH OH (3) 0^ O^H OH OH

H H

lndole-3-glycerol (5‘-Phosphoribulosyl)- phosphate anthranilic acid (enot form )

@ ^ Phosphoqlyceraldebyde Tz HOCH^—C — COOH NHg ^ ^ — COOH

(I) Anthraniiate phosphoribosyltransferase; (2) Phosphoribosyl anthraniiate isomerase; (3) Indole-3-glycerol phosphate synthase (4) Tryptophan synthase (Reference: Luckner, 1990 a)

The biosynthesis of tryptophan, an intermediate in the Shikimate / Chorismate

55 pathway (Figure 1-4) was elucidated to a large extent by employing mutants of Escherichia coli (Davis, 1950). The enzymes involved in this pathway were subsequently identified, isolated and characterised from extracts of cell cultures of Daucus carota (Widholm, 1973). The biosynthesis of tryptophan is a 5 step procedure commencing with the conversion of chorismate to anthraniiate by the action of anthraniiate synthase, as shown in Figure 1-5. Tryptophan synthase the last enzyme in the biosynthetic pathway has also been demonstrated in peas and tobacco (Delmer and Mills, 1968; Chen and Boll, 1971 a,b; Nagao and Moore, 1972). Crespi-Perellino et aL, (1986 a,b) proposed that tryptophan was converted to canthin-6-one, via one of two alternative routes: (1) Tryptophan —> p-carboline-1-propionic acid -» 4,5-dihydrocanthin-6-one —> canthin- 6-one. (2) Tryptophan -» p-carboline-1-propionic acid —> p-carboline-1 -acrylic acid —> canthin- 6-one. In large scale experiments, (DL)-[methylene '"‘C]- tryptophan and (L)- [methylene '‘^C]- tryptophan were fed to cell cultures of A. altissima and the alkaloids were subsequently extracted and isolated. This group demonstrated that incorporation of the '"‘C-label into the alkaloids occurred as follows: 70 % of the ’"^C-label occurred in canthin-6-one which was the major alkaloid constituting 70 % of the total alkaloid fraction. The rest of the ‘"^C-label was distributed among minor alkaloids 4- hydroxycanthin-6-one; 5-hydroxycanthin-6-one; 4,5-dihydrocanthin-6-one; and canthin- 6-one-3N-oxide. The existence of the ‘"‘C-label in 4,5-dihydrocanthin-6-one showed that p-carboline-1-acrylic acid was not involved in the formation of canthin-6-one alkaloids. The proposed route via 4,5-dihydrocanthin-6-one (Figure 1 -6) was further confirmed by studies in which labelled p-carboline-1 -propionic acid and 4,5- dihydrocanthin-6-one were fed to suspension cultures of A. altissima. This resulted in the labelling of the extracted and isolated canthin-6-one alkaloid, thereby confirming that p-carboline-1-propionic acid and 4,5-dihydrocanthin-6-one are intermediates in the biosynthesis of canthin-6-ones.

56 FIGURE 1-6 The Proposed Biosynthetic Pathway for the Production of Canthin- 6-one Alkaloids in A. altissima Cell Suspension Cultures

Tryptophan -Carboline-1- COOH propionic acid 4 ,5-dihydrocanthin- 6-one

2-hydroxycanthin- OH 10, 6-one 5-hydroxycanthin- 9 6-one

Canthin-6-one

Canthin-6-one- 3N-oxide 4-hydroxycanthin- 6-one

l-hydroxycanthin-6-one

l-methoxycanthin-6-one 1-methoxycanthin- 3-N-oxide

57 Anderson et al. (1986) and Hay (1987) carried out experiments involving the feeding of L-tryptophan and L-(methylene''^C)-tryptophan to suspension cultures of A. altissima that produced 1-methoxycanthin-6-one as the major constituent with canthin-6-one and 1-hydroxycanthin-6-one as the only minor constituents. These experiments showed that feeding large quantities (250 - 500 mg . 1') of L-tryptophan to suspension cultures of A. altissima, resulted in a rapid uptake of L-tryptophan by the cells. Alkaloid production was also enhanced by 67 -76 %, with maximum enhancement occurring when unlabelled tryptophan was fed during the lag phrase (0-7 days) (Anderson et aL, 1986). When L-(methy 1 ine'"^C)-tryptophan was fed into cell cultures of A. altissima at day 0 of the growth cycle, it was well incorporated into canthin-6-one, 1-hydroxy- and 1 -methoxycanthin-6-one. The involvement of tryptamine in the biosynthesis of canthin-6-one alkaloids was also investigated by carrying out feeding experiments with '"^C-tryptamine and L- tryptamine. Crespi-Perellino et al. (1986 a,b) reported lack of incorporation of the '"^C-label of '"^C-tryptamine into the canthin-6-one alkaloids of A. altissima. Hay (1987), however demonstrated that feeding (side chain-2-"‘C)-tryptamine into suspension cultures of A. altissima resulted in a rapid incorporation of the ‘"^C-label into the canthin-6-one alkaloids. Hay (1987) also demonstrated that feeding unlabelled tryptamine at levels of 100 mg . 1% during the lag phrase resulted in an increase in alkaloid production by 17 - 24 %. Subsequent experiments carried out more recently have shown that tryptamine is not incorporated into the canthin-6-one alkaloids, nor were significant levels of the key enzyme responsible for the conversion of tryptophan to tryptamine (tryptophan decarboxylase) found in cell cultures of A. altissima (Yeoman, 1993).

1.2.4.1.2 The Role of a-Ketoglutarate in the Biosynthesis of Canthin-6-one Alkaloids Crespi-Perellino et al. (1986 b) suggested an origin from the tricarboxylic acid cycle (TCA) for the carbon atoms C-3, C-4, C-5 and C-6 of canthin-6-one. They also proposed that P-carboline-1-propionic acid is an intermediate in canthin-6-one alkaloid

58 biosynthesis. Aragozzini et al. (1988), investigated the involvement of p-carboline-1- propionic acid in canthin-6-one alkaloid biosynthesis as well as the origin of the C-3 to C-6 atoms of canthin-6-one. The group of Aragozzini et al. (1988), fed (l-'^C), (2- '^C) and (1,2-'^C) sodium acetate to cell cultures of A. altissima and evaluated the sites of enrichment by '^C-nmr spectrometry. These experiments demonstrated a labelling pattern of canthin-6-one, that is consistent with the involvement of a- ketoglutarate as an intact precursor (Figure 1-7). When (l-'^C)-acetate (CH^^^COOH) was fed into the cultures, it resulted in isotopic enrichment only at C-6 of canthin-6-one, whilst in a parallel experiment with (2-'^C)-acetate ('^CH^COOH) there was isotopic enrichment at C-3, C-4 and C-5. Finally, feeding (l,2-'^C)-acetate ('^CHg-'^COOH), gave enrichment at all four carbon atoms and the presence of one pair of coupled '^C satellites for the C-5 and C-6 signals reflects the likelihood that there was incorporation of only one intact acetate unit into canthin-6-one. If the fate of the acetate carbons in the TCA cycle is taken into consideration (Turner, 1971), in addition to the labelling pattern observed in these experiments; it is possible to rule out the involvement of a 4 carbon dicarboxylic acid like succinate, as a precursor of the C-3 to C-6 portion of the canthin-6-one molecule. The labelling pattern obtained in the C-3 to C-6 fragment of canthin-6-one strongly suggests that canthin-6-one is derived from the incorporation of an intact molecule of acetate derived ketoglutarate, with loss of C-1 i.e. the carboxyl group during or after the condensation step and the intermediate formation of p-carboline-1-propionic acid. It is believed that in actual fact two intact acetate units are incorporated, as shown in Figure 1 -7, however only one of these is present in canthin-6-one, as a result of the decarboxylation step. A similar biosynthetic route has been proposed for barman alkaloids from Passiflora edulis and Eleagnus angustifolia, which involved the condensation of the keto group of a ketoacid, pyruvate with the amino group of tryptophan and the subsequent loss of the carboxyl group of pyruvate (Herbert & Mann, 1982).

59 FIGURE 1-7 The Role of a-Ketoglutarate in Canthin-6-one Alkaloid Biosynthesis in Cell Suspension Cultures of A. altissima

CO OM

NH, COOH COOK MOOC h OOC h OOC

(1) Tryptophan; (2) a-Ketoglutarate; (3) Possible intermediate; (4) (3-CarboIine-l- propionic acid; (5) Canthin-6-one. (Reference: Aragozzini el al., 1988).

1.2.4.1.3 The Methylating Ability of A. altissima Cell Suspension Cultures Hydroxylated canthin-6-ones occur commonly in species of the Simaroubaceae (Liu et al., 1990 a,b; Simao et ai, 1991) as well as A. altissima (Crespi-Perellino et aL, 1986 a,b). Hydroxylation at any of the carbons of canthin-6-one, could be followed by méthylation, leading to a wide range of structural types of canthin-6-one alkaloids. This consequently increases the potential for producing biologically active compounds. Evidence presently exists to support the fact that L-methionine in its active form, S-adenosylmethionine, is the major donor of 0- and N- methyl groups in plants (Roberts, 1974 a,b). When L-(methyl*'’C)-methionine was fed to cell suspension cultures of A. altissima, incorporation of the ^^C-label into l-methoxycanthin-6-one occurred (Anderson et aL, 1987 b). Maximum incorporation of 2 to 3 % was observed when L-(methyl‘‘*C)-methionine was fed to cultures in the late exponential phase i.e. days 27-29 of the growth cycle. L-(methyl'^C)-methionine fed to cell suspension cultures of A. altissima, resulted in the production of l-methoxycanthin-6-one which showed isotopic enhancement at 56.75 ppm, corresponding to the methyl carbon. This observation confirms the involvement of L-methionine in the méthylation of 1-

60 hydroxycanthin- 6-one. (Anderson et aL, 1987 b). Cell cultures developed by Anderson et al. (1983, 1986, 1987 a,b), produced a cell line with 1-methoxycanthin- 6-one as the major constituent, and canthin- 6-one and 1 -hydroxycanthin- 6-one as minor constituents. Time course studies carried out by Roberts et al. (1989), showed that 1- hydroxycanthin- 6-one methyltransferase activity was present throughout the growth cycle of A. altissima cell suspension cultures. This activity remained high throughout most of the lag and exponential phases i.e. (days 0 to 28), thus suggesting that the availability of the substrate l-hydroxycanthin- 6-one may be the limiting factor in cultures which exhibit low levels of l-methoxycanthin- 6-one. Further purification of cell free extracts of A. altissima cell suspension cultures containing hydroxycanthin- 6-one methyltransferase activity, demonstrated the presence of enzymes capable of methylating 1-, 8- and lO-hydroxycanthin- 6-one. The ability of cell free extracts to produce 8 and lO-methoxycanthin- 6-one, not previously reported as occurring in cell cultures of A. altissima, is interesting since 10- hydroxycanthin- 6-one and its methylated form have not been previously isolated from A. altissima and 8-hydroxycanthin- 6-one has only been reported as occurring in A. exelsa (Cordell et al., 1978; Roberts, 1991). The methylating capability of these cultures suggests that the variety of methylating products produced by these cultures is determined by the availability of the appropriate hydroxycanthin- 6-one.

1.2.4.2 Plant Cell Culture in the Study of Coumarin Biosynthesis Most of the previous work done on the biosynthesis of coumarins has mainly been by tracer techniques (Brown, 1960; Brown et al., 1988; Dewick, 1985; Dewick, 1990), but plant cell culture techniques could be a better alternative. With plant cell culture techniques, the enzymes involved in the these pathways can easily be identified, isolated and characterised. Since plant cell cultures of A. altissima produce significant levels of the methylated coumarins scopoletin and isofraxidin (Hay, 1987; Roberts, 1991), they are a potentially useful system for studying the biosynthetic pathway for the formation of these methylated coumarins. Feeding cultures of A.

61 altissima with L-[methyl '"‘C] methionine resulted in incorporation of the methyl ‘"Re label into the methylated coumarin scopoletin at levels 0.8 %; consequently indicating the existence of coumarin methyltransferase enzyme activity in cell cultures of A. altissima (Anderson et al., 1987 b). Coumarins are diverse types of benzopyrone derivatives. They occur, for example, as 7-oxygenated coumarins, furanocoumarins, 3-(dimethylallyl) coumarins, pyranocoumarins and phenylcoumarins. They have also been found to occur as esters and glucosides of previously known phenols or alcohols. Well over a thousand coumarins are known to occur in nature and many hundreds more have been synthesised in the laboratory. They have applications ranging from use as anticoagulant drugs to use as laser dyes and fluorescent brighteners (Murray, 1989). Most simple coumarins are of two fundamental types: (1) Those lacking oxygenation at C-1 para to the propanoid side chain (there are less than 50 examples of this category which includes coumarin) (2) Those bearing oxygenation at C-1 para to the propanoid side chain e.g. umbelliferone. Brown (1960), proposed and confirmed on the basis of tracer investigations that transcinnamic acid functions in appropriate plants as the common precursor of all coumarins and that its ortho-hydroxylation leads to the elaboration of coumarin and it’s para-hydroxylation leads to 7-hydroxycoumarin. It was established very early in biosynthetic investigations that coumarin is derived from shikimic acid via phenylalanine and cinnamic acid (Kosuge and Conn, 1959; Stoker and Beilis, 1962).

1.2.4.2.1 Biosynthesis of Coumarins from L-Phenylalanine L-Phenylalanine is the precursor of coumarins, flavonoids and lignin in plants as shown in Figure 1-4. L-Phenylalanine is formed from chorismate via prephenate as shown in Figure 1-8. This pathway which has been elucidated in plants and microorganisms (Luckner, 1990 b), involves 3 enzyme catalysed steps: the conversion

62 of chorismate to prephenate, which is finally converted to L-phenyialanine via phenylpyruvate as shown in Figure 1-8.

FIGURE 1-8 The Biosynthesis of L-Phenylalanine from Chorismate

COOH COOH COOH CO CO H j N - C - H

COOH HOOC CH, ÇH, ÇH;

0 ^ ■^COOH 6 h ÔH Chorismic acid Prephenic acid phenylpyruvic L-Phenyl- acid alanine

(1) Chorismate mutase, (2) Prephenate dehydratase, (3) Phenylalanine aminotransferase (Reference: Luckner, 1990 b).

Simple coumarins from higher plants are almost always derived from cinnamic acid, which is formed from phenylalanine by the action of the enzyme Phenylalanine ammonium-lyase (PAL) (Dewick, 1991).

I.2.4.2.2 Biosynthesis of Coumarins Lacking Oxygenation at the 7-Position The biosynthesis of coumarins lacking oxygenation at the 7-position, as exemplified by coumarin, is shown in Figure 1-9. Kosuge and Conn (1959) proposed that the conversion of trans-cinnamic acid to coumarin shown in Figure 1-9, involves ortho-hydroxylation and glucoside formation at the 2 -hydroxyl position, to form coumarinyl glucoside or 2 - glucosyloxycinnamic acid. They further proposed that isomérisation to the cis isomer, which has been known to occur in plants (Roberts and Link, 1937), takes place when 2 -glucosyloxycinnamic acid is hydrolysed in vitro to the unstable cis -2 - hydroxycinnamic acid (coumarinic acid), which subsequently undergoes spontaneous

63 lactonisation to form the free coumarin. Kosuge and Conn (1959) and later Stoker and Beilis (1962) demonstrated the formation of trans -2 -hydroxycinnamic acid and its glucoside from transcinnamic acid by Melilotus alba in vivo. Gestetner and Conn (1974), subsequently identified a chloroplast enzyme in the same species mediating ortho-hydroxylation. Kleinhoff et al. (1967) demonstrated the glucosylation of trans-2 -hydoxycinnamic acid in cell free extracts of M. alba by a glucosylating enzyme. The enzyme p-glucosidase which specifically hydrolyses the cis-glucoside, releasing coumarin, has been discovered in M. alba leaves (Kosuge and Conn, 1961). It is now agreed that trans-cis isomérisation, constituting the final step in the formation of coumarinyl glucoside is light, rather than enzyme catalysed. Haskins et al. (1964) concluded that, since neither steaming of M. alba leaflets nor maintenance at low temperatures greatly affected their ability to bring about trans-cis conversion, the reaction could not be enzyme mediated. They subsequently presented evidence that it was, in fact, a photochemical reaction, effected by wavelengths below 360 nm.

FIGURE 1-9 The Biosynthesis of Coumarins Lacking Oxygenation at the 7-Position e.g. Coumarin

Phenyl alanine

PAL(Phenylalanine ammonium lyase)

C H = C H - C O O H C H = C H - C O O H C H = C H - C O O H

OH 0 - g l u c o s y 1

trans-cinnam ic trans-2 -hydroxy c o u m a r i n y l a c i d cinnam ic acid g l u c o s i d e

c o u m a r i n

64 Stoker and Beilis (1962), also demonstrated that the cis acid is an effective coumarin precursor. They therefore suggested that, alternatively coumarin and coumarinyl glucoside may arise from a common precursor by independent pathways. They suggested a pathway from trans-cinnamic acid to coumarin via cis-cinnamic acid.

1.2.4.2.3 Biosynthesis of 7-Oxygenated Coumarins The biosynthesis of 7-oxygenated coumarins, as exemplified by umbelliferone is depicted in Figure 1-10. The first reaction in this pathway is the 4 -hydroxylation of cinnamic acid. This reaction is common to the biosynthesis of a number of secondary plant products, most notably lignin. The enzyme involved is a cytochrome P^^g-dependent mono oxygenase which specifically utilises NADPH as a reducing agent. Such reactions have been shown, in vitro, to proceed rapidly and without the release of intermediates (Hamerski et aL, 1990). The formation of umbelliferone (7-hydroxycoumarin) and hemiarin (7- methoxycoumarin) from cinnamic acid were found to occur via 4 -hydroxycinnamic acid (Brown et aL, 1964; Austin and Meyers, 1965 a). This investigation was carried out by in vivo tracer techniques involving Hydrangea macrophylla The conversion of 4 -hydroxycinnamic acid to umbelliferone involves a 2 - hydroxylase acting on 4 -hydroxycinnamic acid. This conversion has been reported to occur in a chloroplast fraction of Hydrangea macrophylla which produces 7- oxygenated coumarins (Brown, 1981). The enzyme hydroxylated cinnamic acid slowly, but rapidly converted 4 -hydroxycinnamic acid to umbelliferone presumably via 2 4 -dihydroxycinnamic acid, as shown in Figure 1-10. Another 2 -hydroxylase from Melilotus alba, reportedly hydroxylated cinnamic acid at a much higher rate, while having a low hydroxylation rate for 4 -hydroxycinnamic acids (Gestetner and Conn, 1974; Brown, 1981). As the existence of cinnamic acid 4 -hydroxylases in plants is assumed to be almost universal, owing to a requirement for this enzyme in lignin synthesis, it was concluded that the presence or absence of one or both of these 2 -hydroxylases controls whether a species produces 7-oxygenated or non-oxygenated

65 coumarins (or, in rare cases, both) (Brown, 1981). As in the case of coumarin, glucoside formation is prominent in the formation of 7-oxygenated simple coumarins (Brown, 1981). Umbelliferone in Hydrangea macrophylla (Austin and Meyers, 1965 b) and Pimpinella magna (Floss and Paikert, 1969) and hemiarin (7-methoxycoumarin) in Lavendula officinalis (Brown, 1965), occur almost entirely in the glucoside form.

FIGURE 1-10 The Biosynthesis of 7-Oxygenated Coumarins e.g. Umbelliferone

Phenyl alanine

PAL(Phenylalanine ammonium lyase)

C H = C H - C O O H C H = C H - C O O H C H = C H - C O O H

HO OH trans-cinnam ic 4 - h y d r o x y 2 ,4 -dihydroxy a c i d cinnam ic acid cinnam ic acid

H O ^ O " ^ 0

U m belliferone

66 1.2.4.2.4 Biosynthesis of di- and tri-Hydroxy Coumarins Mono-hydroxycoumarins e.g. umbelliferone, are clearly derived from cinnamic acids of corresponding oxygenation patterns. Attempts to demonstrate biosynthesis of the ortho-dihydroxycoumarins e.g. aesculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) from caffeic acid were however not successful (Sato and Hasegawa, 1972). These findings and the fact that umbelliferone has been reported to be a precursor of (i) the 7,8-dihydroxycoumarin, daphnetin (Dewick, 1988), and (ii) coumarins with hydroxy or methoxy substituents at positions 6, 7 and 8 e.g. fraxetin and isofraxidin (Dewick, 1990), suggested that additional oxygenation of the nucleus may sometimes occur in simple coumarins since it occurs routinely in furanocoumarins (Brown, 1981). A series of feeding experiments have been conducted to investigate the biosynthesis of daphnetin (7,8-dihydroxycoumarin) (Figure 1-15), using shoots of Daphne mezerum (Dewick, 1988). Umbelliferone was incorporated more efficiently than p-coumaric acid (4 -hydroxycinnamic acid) and caffeic acid was poorly utilised. These findings support the idea of umbelliferone being a general precursor of other plant coumarins that bear further oxygen substituents on the aromatic ring e.g. daphnetin, rather than the hypothesis that additional substitution occurs at the cinnamic acid stage (Figure 1-11) (Dewick, 1988).

FIGURE 1-11 The Formation of Daphnetin from Umbelliferone

HO 0 ^ 0

Umbelliferone Daphnetin

67 1.2.4.2.5 Biosynthesis of Methylated Coumarins: Scopoletin, Fraxetin, Isofraxidin and Puberulin Previous work suggested that scopoletin like the monohydroxycoumarins e.g. umbelliferone (Figure 1-10) was biosynthesised from phenylalanine and cinnamic acid via p-coumaric acid (4 -hydroxycinnamic acid). Caffeic acid (3,4-dihydroxycinnamic acid) and ferulic acid (3-methoxy-4-hydroxycinnamic acid) were also suggested as intermediates in scopoletin biosynthesis (Fritig et aL, 1970; Loewenberg, 1970). However, results obtained from more recent work is consistent with umbelliferone being the precursor of both scopoletin and isofraxidin (Dewick, 1990). Brown (1981) also reported that umbelliferone was the probable precursor of Daphnetin (Figure 1 - 11 ). This hypothesis was further strengthened by studying the sequence of steps leading to the biosynthesis of the dimethylallyloxycoumarin, puberulin (Figure 1-12), which were investigated through feeding experiments in Agathosma puberula (Brown, et aL, 1988; Dewick, 1985, 1990). The labelled coumarins umbelliferone, aesculetin, scopoletin and isofraxidin were well incorporated and the pathway is suggested to occur as shown in Figure 1-12 (Dewick, 1990). Although fraxetin was not tested as a precursor, its demonstrated presence in A. puberula supports its role as an intermediate. This series of steps strongly support aesculetin as the precursor of scopoletin; which subsequently serves as a precursor of fraxetin (Figure 1-12), which is then converted to isofraxidin (as shown in Figure 1-12). Dewick (1985) reported that when ferulic, sinapic and caffeic acids were fed to Agathosma puberula and levels of incorporation compared with those of p-coumaric acid, umbelliferone and scopoletin, the results showed that ferulic, sinapic and caffeic acid were all poorly utilised in comparison to p-coumaric acid, while the coumarins umbelliferone and scopoletin were well incorporated. On the basis of these results, the pathway shown in Figure 1-12 was proposed for the biosynthesis of puberulin via scopoletin. The sequence of steps also suggests that the first occurrence of méthylation in this pathway is at the aesculetin —> scopoletin step in this biosynthetic pathway.

68 FIGURE 1-12 The Biosynthesis of Scopoletin, Fraxetin, Isofraxidin and Puberulin

COOH

HO 0 ^ 0 P-Coumaric Umbelliferone acid

HO " O ^ O

Aesculetin

CH3 O

HO " ^ 0 " ^ 0

Scopoletin

CHtO

0 ^ 0

OH Fraxetin

Isofraxidin

Puberulin

69 1.3 Methyltransferases in Plants Transmethylation reactions involve the transfer of one carbon unit in the form of a methyl group between two substrates. The generation and utilisation of methyl groups is a very important aspect of the metabolism of all cells, playing a particularly important role in the biosynthesis of secondary metabolites. Methyltransferases are the enzymes responsible for catalysing transmethylation reactions. They catalyse O- and N-methylation reactions which are usually involved in the biosynthetic pathway for the generation of secondary metabolites in plants animals and microorganisms. In order to elucidate the detailed pathways of secondary metabolite biosynthesis it is necessary to demonstrate the presence of enzymes responsible for the various biosynthetic steps. The presence of methyltransferase enzymes have been demonstrated in a variety of biological tissues and several methyltransferases obtained from plant sources have been identified, isolated and characterised. The occurrence of a vast multitude of secondary plant products possessing one or more methyl groups in their structure, is an indication of the frequency and importance of transmethylation reactions in this area of plant metabolism. As in many other natural products N- and O-methyl groups are of common occurrence in alkaloids. In many cases, it has been demonstrated that these methyl groups are derived from L-methionine as demonstrated for nicotine (Dewey et aL, 1954; Agurell et aL, 1967). Evidence now exists to support the fact that SAM is the immediate methyl donor in these transmethylation reactions (Mann and Mudd, 1963; Poulton, 1981). The subsequent isolation of several SAM dependent methyltransferases have confirmed the involvement of SAM in these méthylation reactions (Suzuki and Takahashi, 1975; Poulton, 1981; Preisig et aL, 1989; Frenzel and Zenk, 1990 a,b; Edwards and Dixon, 1991; Hibi et aL, 1992). Méthylation reactions fulfil many physiological functions in plants. The méthylation of polyphenol ic compounds is known to reduce the chemical reactivity of phenolic groups (Poulton, 1981). O-Methylation is also known to increase the lipid solubility and volatility of polyphenol ic compounds. Méthylation could also be

70 responsible for possible changes in absorption spectra which occurs on méthylation of anthocyanins. This could be an important factor in attracting or deterring pollination vectors (Poulton, 1981). Méthylation reactions have also been reported to play an important role in the biosynthesis of lignin. Lignin an amorphous heteropolymer is formed by the random oxidative copolymerization of substituted cinnamyl alcohols i.e. p-coumaryl, coniferyl and sinapyl alcohols. Two transmethylation reactions involving SAM occur during lignin biosynthesis. Caffeic acid is methylated to ferulic acid which is thought to be hydroxylated in vivo to 5-hydroxyferulic acid. Subsequent méthylation of this intermediate at the 5-position yields sinapic acid (Poulton, 1981). Byerrum et al. ( 1954), using double labelled methionine, successfully demonstrated the origin of the methylester groups of lignin. In other experiments Hess (1964) and Shimada and Higuchi (1970), isolated methyl labelled ferulate and sinapate from several plant species following the administration of [CH^-'^^CJ-methionine. The discovery of catechol-O-methyltransferases which methylate caffeic acid specifically at the meta position in apple cambium tissue (Finkle and Nelson, 1963) and pampas grass (Finkle and Masri, 1964) represented a very significant advance towards a greater understanding of lignin biosynthesis. Since then further enzymes of this type have been isolated whose enzymological properties are in agreement with their involvement in the lignification process. Lignin was found deposited around wounds in wheat leaves infected with the non-pathogenic fungus Botrytis cinerea. This was preceded by increases in PAL, caffeate and 5-hydroxyferulate-methylating activities, specifically within the lignifying tissues (Maule and Ride, 1976). Comparable data have been obtained following the infection of tuber slices of potatoes by Phytophthora infestans (Friend and Thornton, 1974). Méthylations also play an important role in transmethylation reactions involved in alkaloid biosynthesis (De Luca et al., 1987; Frenzel and Zenk, 1990 a,b; Rueffer et al., 1990). Alkaloids have been the focus of extensive research because of the important pharmacological properties they exhibit. The biosyntheses of various

71 alkaloids have been widely studied by isotope incorporation and the isolation of enzymes and intermediates involved in the pathways (Poulton, 1981).

1.3.1 O-Methyltransferases in Plants O-Methylation plays an important role in secondary metabolism in plants. O-methylation is a frequently occurring step in the biosynthesis of plant phenolic compounds, including o-dihydric phenols and certain polyphenolic compounds (Tsang and Ibrahim, 1979). Several SAM dependent 0-methyltransferase (O-MT) enzymes have been identified in the metabolism of lignin, flavonoid compounds, phenylpropanoids, furanocoumarins and alkaloids. Various studies involving the extraction, isolation, purification and characterisation of 0-methyltransferases have been reported in literature. The elucidation of detailed biosynthetic pathways for the formation of secondary metabolites depends on these studies. The characteristics of the 0-methyltransferases described in this section are summarised in Table 1-14.

1.3.1.1 0-Methylation of Caffeic acid in Lignin and Flavonoid Biosynthesis

CH=CH-COOH CH=CH-COOH

HO SAM SAH OCH

Caffeic acid Ferulic acid

Caffeic acid 0-methyltransferase (O-MT), catalyses the 3-O-methylation of

72 caffeic acid (3,4-dihydroxycinnamic acid) to yield ferulic acid (3-methoxy-4- hydroxycinnamic acid). This enzyme has been isolated and purified from a number of plant sources including elicited cell suspension cultures of Medicago sativa from which caffeic acid O-MT was purified to homogeneity (Edwards and Dixon, 1991). This enzyme which exists as two isomers (i and ii) was found to be highly specific for the 3-hydroxy group of substituted cinnamic acids. Other sources of the enzyme include; the leaves of beet (Beta vulgaris) from which caffeic acid O-MT was partially purified (Poulton and Butt, 1975); and tobacco cell suspension cultures (Tsang and Ibrahim, 1979). The O-MT enzyme from tobacco cell suspension cultures was named O- dihydricphenol O-MT because it catalysed the O-methylation of a wide range of phenolic substrates including cinnamic acids; 5-hydroxyferulic acid and 3,4,5-tri- hydroxycinnamic acid; the coumarins, daphnetin and aesculetin and some flavonoids (Tsang and Ibrahim, 1979). Caffeic acid, 5-hydroxyferulic acid and quercetin are the best substrates for this enzyme. Caffeic acid was the best substrate for isomer i and quercetin for isomer ii. The méthylation of caffeic acid to ferulic acid shown above, is believed to be an intermediate stage in the biosynthesis of lignin and some flavonoids (Finkle and Nelson, 1963; Finkle and Masri, 1964; Poulton, 1981). Kuhnl et aL (1989), also reported the presence of an O-MT in cell suspension cultures of Daucus carota which specifically converts caffeoyl-CoA to feruloyl-CoA.

1.3.1.2 O-Methylation of Flavonoid Compounds

1.3.1.2.1 4 -0-Methylation of Isoflavones in Isoflavone Biosynthesis A highly specific 0-methyltransferase catalysing the transfer of the S-methyl group of SAM to the 4'-hydroxyl group of isoflavones has been detected in seedlings and cell suspension cultures of chick pea (Cicer arietinum) (Wegenmayer et aL, 1974). It was subsequently isolated and characterised and given the systematic name SAM: isoflavone 4'-0-MT, since it is specific for the méthylation of 4'-hydroxy

73 flavones including: the méthylation of diadzein (7,4'-dihydroxy isoflavone) to formomonetin (7-hydroxy,4'-methoxyisoflavone) or the méthylation of genistein (7,5,4'-trihydroxy isoflavone) to biochanin A (7,5-dihydroxy,4'-methoxyisoflavone) as shown below.

I] SAM SAH R 0 OCH

Diadzein, R = H Formomonetin, R = H Genistein, R = OH Biochanin A, R = OH

This enzyme is believed to be responsible for catalysing the last step in the biosynthesis of isoflavones since it demonstrated a high degree of specificity and failed to methylate two intermediates in isoflavone biosynthesis i.e. p-(hydroxy)- cinnamic acid and 5,7,4'-trihydroxy flavanone (naringenin) (Wegenmayer etal.^ 1974).

1.3.1.2.2 5-0-Methylation of Isoflavones An isoflavone 5-0-methyltransferase has been partially purified and characterised from the roots of yellow lupin (Lupinus luteus). This enzyme specifically mediates the transfer of methyl groups from SAM to the 5-hydroxy position of isoflavones as shown below. The activity of this enzyme accounts for the presence of a number of naturally occurring 5-O-jmethyl flavones in IL. luteus roots. Some of those identified include: 5-0-methylgenistein, 5-0- methylderrone, 5-0-methyl-2^-hydroxygenistein and 5-0-methyl-2'-hydroxyderrone (Khouri et at., 1988 b).

Apart from the anthraquinone glucosyltransferase system of Cinchona

74 succirubra cell cultures which glucosylated the 5-position of flavonoids (Khouri and Ibrahim, 1987), this is the only other reported instance of an enzyme which acts on position 5 of isoflavonoids.

SAM SAH OH 0

Genistein 5-0-Methylgenistein

SAM SAH OH 0

Derrone 5-0-Methylderrone

Although the 5-hydroxyl group of flavonoids is the least reactive position, due to its chelation with the carbonyl group, it seems possible that the activity of the

75 isoflavone 5-0-methyltxansferase may be due to the specific binding of the flavonoid substrate and the cosubstrate in a geometrical arrangement relative to each other, such that méthylation at the 5-position is preferentially catalysed (Khouri et al., 1988 b).

1.3.1.2.3 0-MethyIation of (+)6a-Hydroxymaackiain in Pisatin Biosynthesis

HO CH3 O 0 > 0 7 ^ SAM SAH (+)6a-Hydroxymaacfciain (-H)-Pisatin

An enzyme which synthesises pisatin by the O-methylation of the 3-hydroxyl group of (+) 6a-hydroxymaackiain was extracted from CuClj-stressed pea seedlings. Many plants respond to microbial infection or other forms of stress by synthesising and accumulating low molecular weight toxic compounds called phytoalexins at the site of challenge (Preisig et al., 1989). The isoflavonoid phytoalexin pisatin is synthesised by Pisum sativum in response to microbial infection and certain other forms of stress. The O-MT enzyme catalyses the terminal step in the synthesis of Pisatin as shown above.

1.3.1.2.4 3'-0-Methylation of Luteolin in Flavone Glucoside Biosynthesis An O-MT enzyme has also been isolated and partially purified from cell suspension cultures of parsley that catalyses the transfer of the S-methyl group of SAM to the meta or 3' position of 0-dihydric phenols, e.g., the conversion of luteolin (5,7,3',4'-hydroxyflavone) to chrysoeriol (5,7,4'-trihydroxy,3'-methoxyflavone (Ebel et al., 1972). Enzyme assays carried out with the partially purified methyltransferase show that only ortho-dihydricphenols can serve as substrates for the enzyme and

76 méthylation took place exclusively in the meta position of T-substituted 3',4'-dihydric phenols.

SAM SAH OH 0 OH 0

Luteolin Chrysoeriol

This O-MT enzyme is thought to be directly related to flavone glucoside biosynthesis. The enzyme's high affinity for luteolin and it’s 7-0-glucoside supports the assumption that one or both of these substrates is the natural precursor for 3 -0- methylated flavone glucosides (Ebel et al., 1972). The enzyme catalyses the 3 -0- methylation of luteolin to chrysoeriol as shown above. The pathway for flavone glucoside biosynthesis that occurs via phenylalanine to 4-coumaroyl-CoA and subsequently to a flavone glucoside (malonylapiin) has been established in irradiated cell suspension cultures of parsley (Petroselinum hortense). Ebel and Hahlbrock (1977), also reported the presence of an 0-methyltransferase which methylates flavones and flavonols specifically at the 3'-position and is thought to be involved in the biosynthesis of flavone glucosides in P. hortense The 3 -0- methyltransferase as well as a glucosyltransferase (Sutter and Grisebach, 1973) were also shown to be specific for the méthylation of various flavonoid intermediates in the inadiated cell cultures of P. hortense (Kreuzaler and Hahlbrock, 1973).

1.3.1.2.5 7-0-Methylation of 3-MethyIquercetin in Flavone Glucoside Biosynthesis An O-MT which catalyses the 7-0-methylation of | 3-methylquercetin to 3,7-

77 dimethylquercetin has been isolated from Chrysosplenum americanum shoot tips (Khouri et al., 1988 a). Chrysosplenum americanum accumulates a number of tri- to penta-O-methylated flavonol glucosides. The enzyme SAM: 3-methylquercetin O-MT, which exhibits a high degree of specificity for 3-methylquercetin, is thought to catalyse the second methyl transfer in the biosynthesis of polymethylated flavonols. The pathway is believed to be: quercetin —> 3-methylquercetin 3,7-dimethylquercetin -> 3,7,4'-trimethylquercetin.

HO OH OH

OCH OCH SAM SAH OH 0

3-Methylquercetin | 3,7-Dimethylquercetin

The O-MT enzymes catalysing the other transmethylation step had been previously demonstrated to occur in Chrysosplenum americanum shoots tips (De Luca and Ibrahim, 1985). A similar enzyme system has been isolated and characterised from Citrus mitis fruit peel, seedling, root and peel cultures (Ibrahim et al., 1982) as well as tobacco cell cultures (Tsang and Ibrahim, 1979).

1.3.1.3 5-0- and 8-0-Methylation of Furanocoumarins Two SAM: furanocoumarin O-MT enzymes have been isolated, purified and characterised from cell suspension cultures of Ruta graveolens (Sharma et al., 1979; Sharma and Brown, 1979). They catalyse the transfer of the S-methyl group of SAM

78 to either the 5- or 8-hydroxyl group of the psoralen nucleus leading to the production of their respective methylated substituents as shown below. Identification of this O-MT system accounts for the existence of a number of furanocoumarins which bear methoxy substituents (Sharma ct a/., 1979).

0 SAM SAH 0 O'^O OCH3

8-Hydroxypsoralen 8-Methylpsoralen

OCH3

0 "0 SAM SAH

5-Hydroxypsoralen 5-Methylpsoralen

1.3.1.4 3-0-Methyiation of Dopamine in the Biosynthesis of Mescaline An O-MT enzyme from Lophophora williamsii (peyote) has been partially purified and characterised. The enzyme catalyses the transfer of the S-methyl group

79 of SAM to the 3-hydroxyl group of dopamine to give 4-hydroxy-3-methoxyphenethylamine (Basmadjian and Paul, 1971). This méthylation reaction is believed to be part of the biosynthetic pathway to mescaline in L. williamsii. The in vivo investigations of the biosynthesis of mescaline have suggested that the 3-0-methylation of dopamine to give 4-hydroxy-3-methoxy phenethylamine, as shown below is the first step in mescaline biosynthesis.

NH2 SAM SAH HO

Dopamine 4-hydroxy-3-methoxyphenethylamine

1.3.1.5 4 - 0 - Méthylation of 3'-Hydroxy-N-methyI-(S)-coclaurine in the Biosynthesis of Reticuline

HO' CH HO CH3

HO HO

HO SAM

3'-Hydroxy-N-methyl-(S)-coclaurine (S)-Reticuline

80 SAM: 3'-hydroxy-N-methyl-(S)-coclaurine-4'-0-MT has been found in cell cultures of several plant species and has been purified from Berheris koetineana cell cultures (Frenzel and Zenk, 1990 b). This enzyme catalyses the 4'-0-methylation of 3'-hydroxy-N-methyl-(S)-coclaurine to form reticuline as shown above. The enzyme demonstrates a high degree of substrate specificity and stereoselectivity. An absolute requirement for the C-4'-methylation reaction is the presence of an adjacent hydroxyl function, and the enzyme recognises only the (S)- enantiomer. This enzyme catalyses the last step in the biosynthesis of (S)-reticuline, the central intermediate in isoquinoline alkaloid metabolism.

81 TABLE 1-14 Properties of 0-Methyltransferases of Plant Origin

Methyltransferase (MT) Source Substrate Product PH Kml K .2 Mwt Stability of Enzyme Ref pM pM xl(P

Caffeic acid-3-O-MT (2 isoforms Medicago sativa cell caffeic acid ferulic acid 7.2 i53 13-15 41 stable for 2 months at -70°C 1 of the same enzyme (i and ii) suspension cultures Ü59 Ü3-15 Caffeic acid-3-O-MT Beta vulgaris leaves caffeic acid feruüc acid 6.5 68 12.5 stable for 1 month at -20“C 2 O-Dihydricphenol-O-MT (2 Tobacco cell suspension i caffeic acid i ferulic acid i7.3 i 100 14 175 3 isoforms of the same enzyme) cultures ii quercetin ii rhamnetin Ü8.3 Ü45 Ü4.4 Ü70 Caffeoyl CoA-3-O-MT Daucus carota cell caffeoyl CoA feruloyl-CoA 7.4 100 30 4 suspension cultures Isoflavone-4'-0-MT (1 enzyme Cicer arietinwn seedlings i diadzein i formomonetin 9.0 80 160 110 stable for several months in lOmM 5 acting on 2 substrates i and ii) & cell suspension cultures ii genistein ii biochanin A DTT + cone (NH4 )2 S0 4 at 4“C O-Dihydrophenicol-O-MT Parsley cell suspension luteolin chrysoeriol 9.7 46 150 48 lost 30% of it’s activity after 1 6 cultures month in Tris/HCl buffer at -20°C 3-Methylquercetin-7-O-MT Chrysosplenum 3-methyl 3,7-dimethyl- stable for 2 months in Tris/HCl 7a oo to americanum shoot tips quercetin quercetin buffer +10mM DTT Isoflavone-5-O-MT Lupinus luteus roots genistein 5-0-methyl 7.0 stable for 2 months in Tris/HCl 7b genistein buffer 4- lOmM DTT 4- 10% glycerol

6a-Hydroxymaakian-3-0-MT Pisum sativum seedlings 6a-hydroxy- 6a-hydroxy-3- 7.9 2.3 35 43 -60 stable for several months at -80°C 8 (CuCI^ stressed) maackian methylmaackian with ImM EDTA4-10mM DTT Furanocoumaiin-O-MT (2 Ruta graveolens cell 5-hydroxypsoralen 5-methylpsoralen 9,10 enzymes acting on 2 substrates) suspension cultures 8-hydroxypsoralen 8-methylpsoralen Dopamine-3 -O-MT Lophophora williamsii dopamine (3,4-di- 4-hydroxy-3- 8.3 175 410 11 hydroxyPhen- methoxy- ±1 ethylamine) phenethylamine 3'-Hydroxy-N-methyl-(S)- Berberis koetiniana cell 3'-hydroxy-N- reticuline 8.3 4.5 30 40 stable for over a year in 20% 12 coclaurine-4'-0-MT cultures methylcoclaurine glycerol at -20®C

Legend: pH = pH optimum; 1 = of substrate; 2 = of SAM; Mwt = Molecular weight in Dalton References(Refs): 1.Edwards and Dixon (1991); 2.Poulton and Butt (1975); 3.Tsang and Ibrahim (1979); 4.Kuhnlet al. (1989); S.Wegenmayeret al. (1974); ô.Ebel et al. (1972); 7a & 7b.Khouri et al. (1988 a and b); 8.Preisiget al. (1989); 9.Sharma et al. (1979); lO.Sharma and Brown (1979); 11.Basmadjian and Paul (1971); 12.Frenzel and Zenk (1990 b). 1.3.2 N-Methyltransferases in Plants A substantial amount of research has been carried out on N-methyltransferase (N-MT) enzymes in plants, and most of this work was done over the last 10 years. Most of these N-methyItransferases are involved in alkaloid biosynthesis since alkaloids containing methylated nitrogen, are widely distributed in plants (Roberts, 1974 b). Isolation and characterisation of these enzymes has been necessary for understanding the biosynthetic pathways leading to production of various alkaloids. The characteristics of the N-methyltransferases described in this section are summarised in Table 1-15.

1.3.2.1 N-Méthylation of the Amine, Putrescine, to N-Methylputrescine in the Biosynthesis of Tropane and Pyridine Alkaloids Putrescine: N-methyltranferase was partially purified and characterised from cultured roots of Hyoscyamus albus which contains hyoscyamine, a tropane alkaloid, as the major alkaloid (Hibi et aL, 1992). The biosynthesis of tropane and pyridine alkaloids is thought to proceed from L-arginine or L-ornithine via the diamine putrescine which is N-methylated to yield N-methylputrescine by putrescine: N-methyltransferase as shown below.

NH2 ^*^2 SAM SA H NH ^^^2 CHo \ Nicotine Putrescine \ N-Methylputrescine \ -N- I CH

0 -Tropyl Tropane alkaloids

83 This enzyme utilises SAM as the methyl group donor. N-methylputrescine is then converted to tropane alkaloids, e.g., hyoscyamine or pyridine alkaloids, e.g., nicotine. Putrescine N-MT efficiently N-methylates amines which have at least 2 amino groups separated by 3 or 4 methylene groups. Putrescine N-MT has also been isolated from tobacco roots, indicative of the involvement of putrescine as an intermediate in nicotine biosynthesis (Mizusaki et al., 1971). Leete (1967), proposed that the formation of N-methylputrescine from putrescine accounts for the biosynthetic route through which the N-methylpyrollidine ring of nicotine is formed. Schiitte et aL (1966), reported the incorporation of N- methylputrescine into the pyrrolidine ring of nicotine, which further lends support to the proposed pathway. It has been proposed that N-methylputrescine is converted to nicotine via 4- methylaminobutanal and N-methylpyrrolinium.

1.3.2.2 N-Methylation of Nicotinic Acid

,c:;^COOH

SAM SAH CH3

Nicotinic acid Trigonelline

A soluble N-MT which catalyses the méthylation of nicotinic acid (Pyridine-3- carboxylic acid) to form its N-methylated product Trigonelline (as shown above), has been detected in protein preparations from cell suspension cultures of soy bean {Glycine max L.). The purified methyltransferase exhibited a remarkable substrate specificity for nicotinic acid. It failed to methy late a series of other pyridine derivatives tested. The enzymatic formation of trigonelline from nicotinic acid has also been studied using crude preparations from Fenugreek {Trigonella foenum graecum).

84 Trigonelline, the N-methyl conjugate of nicotinic acid is a well known constituent of different plant species and numerous cell cultures. It is considered to be a storage form of nicotinic acid. Conjugation reactions like this play an important role in plant metabolism as they allow storage, detoxification and metabolic regulation of primary and secondary constituents of plant cells (Lynn et aL, 1984; Upmeier et aL, 1988).

1.3.2.3 N-Methylation of Anthranilic Acid

SAM " s A H

Anthranilic acid N-Methylanthranilic acid

An enzyme which catalyses the N-methylation of anthranilic acid to form N- methylanthranilic acid, has been identified, isolated and purified from callus and cell suspension cultures of Ruta graveolens. This méthylation is thought to be the first specific step in rutacridone biosynthesis. Rutacridone, a dihydrofuroacridone alkaloid is one of a number of acridones isolated from the intact plant and tissue cultures of Ruta graveolens L. (Rutaceae) (Baumert et aL, 1983). The occurrence of this group of alkaloids is confined to the Rutaceae.

1.3.2.4 N-Methylation of Xanthines to Yield Caffeine Cell free extracts from unripe green fruits of coffee (Coffea arabica) catalysed the transfer of methyl groups from ‘"‘C labelled SAM to 7-methylxanthosine and 7- methylxanthine, producing theobromine (3,7-dimethyLxanthine), which is further methylated to yield caffeine (Roberts and Waller, 1979). The proposed biosynthetic pathway for the formation of caffeine in Coffea arabica as shown below is from:

85 7-methylxanthosine (starting material) —> 7-methyIxanthine —> 3, 7-dimethyxanthine (theobromine) —> caffeine (1,3,7 trimethylxanthine). This N-MT has been shown to mediate both méthylation steps.

0 ÇH3

N RiboSG Ri boSG

7-Methylxanthosine 7-Methylxanthine rSAM

IS^SAH SAH SAM 0 CH- I - - W N

CH3 0 "N CH3 Caffeine 3,7-Dimethylxanthine

An alternative route for the formation of caffeine in coffee was proposed by Ogutuga and Northcote (1970), who suggested that guanine rather than 7- methylxanthosine was the starting material in caffeine biosynthesis. They proposed the following biosynthetic pathway: guanine -> 7-methylguanine —> 7-methylguanylic acid -> 7-methylguanosine -> 7-methylxanthosine -> 7-methylxanthine -> 3,7- dimethylxanthine Caffeine. Cell free extracts from tea leaves also catalysed the production of caffeine through the méthylation of 7-methylxanthine and theobromine, with SAM functioning as methyl group donor (Suzuki and Takahashi, 1975). This enzyme showed properties similar to the enzyme obtained from coffee fruits.

86 1.3.2.5 N-Methylation of Alkaloids

1.3.2.5.1 N-Methylation of Coniine to N-Methylconiine

N CH3

Coniine N-Methylconiine

An enzyme catalysing the formation of the hemlock alkaloid, methylconiine from coniine (as shown above), and designated coniine-N-methyltransferase has been isolated from Coniiun maculatum. The proposed biosynthetic pathway for hemlock alkaloids is via 5-keto-octanal which is converted to y-coniceine by a transaminase using L-alanine as the amino group donor, y-Coniceine is then reduced to coniine in a reversible reaction followed by the méthylation of coniine to yield methylconiine (Roberts, 1974 b).

1.3.2.5.2 N-Methylation of Cytisine in Quinolizidine Alkaloid Biosynthesis

CH3 NH

SAM SAH 0

Cytisine N-Methylcytisine

87 Wink (1984), reported the occurrence of a SAM: cytisine N-methyltransferase in plants of Laburnum anagyroides and in tissue cultures of Cytisus canariensis and subsequently extracted, purified and characterised the enzyme. This enzyme specifically catalyses the méthylation of cytisine to N-methylcytisine, using SAM as the methyl group donor, as shown above. Cytisine a quinolizidine alkaloid, is thought to be derived from lupanine via 5,6-dehydrolupanine, anagyrine, and rhombifoline as intermediates (Wink and Hartman, 1981). The biogenetic sequence involved in the formation of lupanin from lysine is thought to be the basic pathway for quinolizidine alkaloid biosynthesis (Wink and Hartman, 1980), since lupanine serves as a precursor for many other quinolizidine alkaloids. This enzyme shows considerable substrate specificity. Other quinolizidine alkaloids, both the methylated and demethylated forms of which are known, e.g., angostifoline and albine, were only converted by 10 - 15 % in comparison to cytisine.

1.3.2.5.3 N-methylation of Hydroxytabersonine in Indole Alkaloid Biosynthesis An N-MT which catalyses the N-methylation, at position 1, of 16-methoxy-3- hydroxy tabersonine to form the N-methylated product desacetoxyvindoline (as shown below) has been reported, in young leaves of Catharanthus roseus.

OCH CO.CH SAM SAH CHo COoCH

16-Methoxy-3-hydroxy tabersonine Desacetoxyvindoline

88 This enzyme which has been partially purified and characterised (De Luca et aL, 1987) catalyses the fourth step in the biosynthesis of vindoline from tabersonine. The isolation and characterisation of the enzyme was instrumental to determining the biosynthetic pathway for vindoline biosynthesis. The lack of activity by this enzyme as well as by the acetyltransferase, which catalyses the last step in vindoline biosynthesis, accounts for the lack of vindoline accumulation in cell suspension cultures of C. roseus.

1.3.2.5.4 N-methylation of (S)-Coclaurine in the Biosynthesis of Reticuline

SAM SAH

(S)-Coclaurine (S)-N-Methylcoclaurine

Three distinctly different isoforms of S AM-(R,S)-tetrahydrobenzylisoquinoline N-methyltranferases have been isolated from cell suspension cultures of Berberis koetineatia. These isoforms were designated N-MT-I, II and HI. These three enzymes exhibited different properties and substrate specificities. N-MT-I showed maximal activity with tetrahydropapaverine as substrate and N-MT-II and III, were most active with coclaurine. The enzyme has a broad substrate specificity and therefore was designated (R,S)-tetrahydrobenzylisoquinoline N-MT. 6-0-methylation of the benzylisoquinolines rendered them more favourable as substrates for the N-MT enzymes as compared with 6-hydroxylated bases (Frenzel and Zenk, 1990 a). With the aid of feeding experiments, isolation of intermediates and in vivo nmr techniques, the pathway leading from L-tyrosine to (S)-reticuline (the central intermediate of isoquinoline alkaloid metabolism in plants was elucidated (Stad 1er and

89 Zenk, 1990). A total of 5 enzymes were involved in this pathway of which 3 were methyltransferases (Stadler et aL, 1987). The N-methylation of (S)-coclaurine to (S)- N-methylcoclaurine is the second méthylation step in this pathway, the first being the 6-O-methylation of (S)-norcoclaurine to yield (S)-coclaurine. The last méthylation step is the 4'-0-methylation of (S)-3'-hydroxy-N-methyI-(S)-coclaurine to (S)- reticuline (Section 1.3.1.5).

L3.2.5.5 N-Methylation of Tetrahydroprotoberberine Alkaloids in Benzylisoquinoline Alkaloid Biosynthesis

R 2 R

R 4 SAM SAH

T etrahydroprotoberberine N-Methyltetrahydroprotoberberine

An enzyme which specifically N-methylates certain (S)- tetrahydroprotoberberine alkaloids to form N-methoxytetrahydroprotoberberines, such as (S)-canadine (R^ R2: O-CHg-O, R3: O-CH3,. R^: OCH3) and (S)-nandinine (R^ R;: O-CH2-O, R3: OH, R4: OCH3), at the expense of SAM has been detected in different species of isoquinoline alkaloid producing plant cell cultures (Rueffer et al., 1990). An N-methyltransferase enzyme designated (S)-Tetrahydroprotoberberine-N- methyl transferase has been identified, isolated and characterised from suspension cultures of Eschscholtzia californica and Cotydalis vaginans. This enzyme catalysed the N-methylation of tetrahydroprotoberberines as shown above, and is thought to

90 occupy a key postition in isoquinoline alkaloid biosynthesis. The enzyme showed an unusually high degree of specificity, requiring a specific substitution pattern on the alkaloidal substrate. It only transfers the SAM methyl group onto the chiral alkaloidal skeleton in a stereospecific cis mode. N-methylation of the tetrahydroprotoberberine alkaloids serves as an important branch point in benzylisoquinoline metabolism. (S)-Tetrahydroprotoberberine alkaloids can either be transformed into protoberberine molecules by enzymatic oxidation of ring C (Amann et aL, 1988), or they can be N-methylated and then serve as precursors for large groups of isoquinoline alkaloids such as benzophenanthridines, protopines and tetrahydrobenzazepines or spirobenzylisoquinolines (Cordell, 1981).

91 TABLE 1-15 Properties of N-Methyltransferases of Plant Origin

Methyltransferase (MT) Source Substrate Product pH K .1 Km 2 Mwt Stability of Enzyme Ref pM pM xlO^ Putrescine N-MT Hyoscyamus albus root putrescine N-methylputrescine 9 277 ± 203 ± 62 ± 1 cultures 10 20 0.7 Putrscine N-MT Tobacco roots putrescine N-methylputrescine 8-9 400 110 60 2 Nicotinic acid N-MT Soy bean cell suspension Nicotinic acid N-methylnicotiitic 8 78 55 3 cultures acid (trigonelline) Anthranilic acid N-MT Ruta graveolens cell anthranilic acid N-methyl 4 cultures anthranilic acid Xanthine N-MT (1 enzyme acting Coffee fruits i 7-methylxanthine i theobromine i 8.5 i200 i 10 no significant loss of activity after 3 5 on 2 substrates i and ii) ii theobromine (3,7 ii caffeine (1,3,7- Ü8.5 ii 70 ii 10 to 4 thawings, when stored in liquid dimethylxanthin) trimethylxanthine) Nj with 8mg.mr' BSA protein Xanthine N-MT (1 enzyme acting Tea leaves i 7-methylxanthine i theobromine i8 .4 i 1000 i2 5 storage at -20®C with P-MCE and 6 on 2 substrates i and ii) ii theobromine ii caffeine Ü8.5 ii 1000 ii 25 glucose resulted in the loss of 95% of enzyme activity in a day VO Coniine MT Conium maculatum fruits coniine N-methylconiine 8.2 1550 7 Cytisine N-MT L.anagyroides plants and cytisine N-methylcytisine 8.5 60 17 8 C .canariensis ceU cultures 16-Methoxy-2,3-dihydro-3- Catharanthus roseus leave 16-methoxy-3- desacetoxy 9 hydroxytabersonine N-MT hydroxy vindoline tabersonine T etrahydrobenzylisoquinoline CeU suspension cultures of coclaurine N-methylcoclaurine i7.4 i 60 10 N-MT (3 isoforms of the same Berberis koetiniana Ü7.2 Ü78 enzyme i, ii and iii) iü6.8 iii 60 S-Tetrahydroprotoberberine cis a Eschscholtzia californica i S-canadine i N-methylcanadine a)i8 a)i6.7 a)i 12 a 78 ± enzyme from both sources a and b 11 N-MT (1 enzyme obtained from 2 ceU suspension cultures ii S-stylopine ii N-methyl- b)i8 Ü3.1 b)i 1.7 10% had a half life of 10 days when sources a and b and acting on 2 b Corydalis vaginans ceU stylopine b)i7.0 b72± stored at 4®C substrates i and ii) suspension cultures Ü4.0 10

Legend; pH = pH optimum; 1 = of substrate; 2 = of SAM; Mwt = Molecular weight in Dalton;L = Labernum\ C = Cytisus, References(Refs): l.Hibi et al. (1992); 2.Mizusaki et al. (1971); 3.Upmeier et al. (1988); 4.Baumert et al. (1983) 5.Roberts and Waller (1979); ô.Suzuki and Takahashi (1975); 7.Roberts (1974 b); 8.Wink (1984); 9.De Luca et al. (1987); lO.Frenzel and Zenk (1990 a); 11. Rueffer et al. (1990). 1.4 Aims and Objectives of the Present Study of Ailanthus altissima The aim of this study is to investigate the méthylation of the hydroxylated canthin- 6-ones and coumarins present in A. altissima cell suspension cultures and to identify, isolate and purify the methyltransferase enzymes responsible for these méthylations. This aim was achieved by setting the following objectives:

1. To carry out time course studies for canthin- 6-one alkaloid production in 2 cell lines of A. altissima, with emphasis on investigating the methylating capacity of these two cell lines, by comparing: a) The levels of méthylation product, l-methoxycanthin- 6-one formed from 1 -hydroxycanthin- 6-one by these cell lines, during the growth cycle. b) The l-hydroxycanthin- 6-one methyltransferase enzyme activity exhibited by these cell lines during the growth cycle. c) The ability of these cell lines to methylate other hydroxylated canthin- 6-ones. 2. To identify the two proposed sequences for coumarin méthylation in two cell lines of A. altissima cell suspension cultures i.e. sequence 1 for the méthylation of aesculetin and sequence 2 for the méthylation of fraxetin by comparing: a) The levels of méthylation products (scopoletin from pathway 1 and isofraxidin» from pathway 2), produced by the cell lines during the growth cycle. b) The aesculetin and fraxetin methyltransferase activities exhibited by these cell lines during the growth cycle. 3. To extract and isolate the methyltransferase enzymes of A. altissima cell suspension cultures and subsequently separate the l-hydroxycanthin- 6-one methyltransferase enzyme (1-HMT) from the coumarin methyltransferase enzyme (CMT). 4. To further purify the 1-HMT enzyme of A. altissima cell suspension cultures to a single protein and to characterise the enzyme. 5. To purify and characterise the CMT enzyme of A. altissima cell suspension cultures.

93 2. MATERIALS AND EXPERIMENTAL METHODS

94 2.1 Development of Ailanthus altissima Cultures

2.1.1 Plant Material

The seeds of Ailanthus altissima were obtained from Brunswick and Euston Squares in London WCl.

2.1.2 Callus Formation A. altissima seeds were imbibed overnight in tap water to facilitate germination. The tap water was subsequently decanted and the seeds sterilised firstly by submersion in 1 % mercuric chloride solution. The seeds were then rinsed with copious amounts of sterile distilled water. This was followed by further sterilisation for 20 minutes in 7.5 % domestos solution. The seeds were subsequently considered to be sterile, and were treated aseptically thereafter. The seeds were rinsed several times in sterile distilled water and then transferred onto moist filter paper, where germination occurred at 25 °C under continuous illumination. The germinated seeds were then placed on Murashige and Skoog solid growth medium (Sub-Section 2.1.4), where callus formation was initiated. The callus cultures were maintained at 25 °C, under continuous illumination and were subsequently subcultured at intervals of 2 to 4 weeks.

2.1.3 Development of Suspension Cultures Callus (5 - 10 g) was aseptically transferred into 40 ml of Murashige and Skoog liquid medium (Sub-Section 2.1.4), in a pre-sterilised conical flask, sealed with aluminium foil. The cultures were maintained under conditions, similar to those previously described for callus cultures, in addition to being continuously agitated at 120 rpm on an orbital shaker (Luckham Rotatest Shaker, R 100, Lukham Ltd. Sussex, England).

95 2.1.4 Preparation of Growth Media

2.1.4.1 Murashige and Skoog Liquid Growth Medium Suspension cultures were maintained in Murashige and Skoog liquid medium, which was prepared from 4.71 g . 1' of Murashige and Skoog basal salts (Imperial laboratories, U.K.) (Murashige and Skoog, 1962), containing: 5 % sucrose (BDH) and the growth hormones, 2,4-dichlorophenoxyacetic acid (Sigma Chemical company Ltd., U.K.) and kinetin (Sigma Chemical company Ltd., U.K.), at levels of 1 mg . 1* and 0.1 mg . r' respectively. The medium pH was adjusted to 5.8 prior to sterilisation by autoclaving.

2.1.4.2 Murashige and Skoog Solid Growth Medium The solid growth medium in which the callus cultures were initiated and maintained is composed of liquid medium (as given in Sub-Section 2.1.4.1), to which 1 % agar had been added prior to sterilisation by autoclaving. The medium was then poured into plates, where it set on cooling.

96 2.2 Phytochemical Investigations of Ailanthus altissima Cultures The canthin- 6-one alkaloid and coumarin content of A. altissima cell cultures were investigated, since these cultures have previously been reported to contain canthin- 6-one alkaloids (Roberts, 1991) and coumarins (Hay, 1987; Roberts, 1991).

2.2.1 Extraction of Secondary Metabolites from Tissue cultures ofA. altissima A. altissima cell suspension cultures were harvested at the appropriate times in the growth cycle of the cells by filtering them through cheese cloth. The cells (2 g) were freeze dried and exhaustively extracted with 2 % HCl in methanol (20 ml). The extract was dried down under reduced pressure and subsequently reconstituted in 18 ml of distilled water. The reconstituted extract was basified with a few drops of 0.88 % ammonium hydroxide solution and the basified extract applied to an Extrelut column which was allowed to stand for 30 minutes for equilibration to take place. The canthin- 6-one alkaloids and the coumarins were then exhaustively eluted from the column with chloroform (3 x 20 ml).

2.2.2 Qualitative and Quantitative Identification of Secondary Metabolites from A. altissima Cultures Reference Compounds In the analysis of secondary metabolites from A. altissima tissue cultures, certain canthin- 6-one alkaloids were used as reference compounds. These include: canthin- 6-one; l-hydroxycanthin- 6-one and 1-methoxycanthin- 6-one. These alkaloids were previously isolated from A. altissima callus and suspension cultures, and were characterised by chromatographic and spectroscopic techniques described in Sub- Sections 2.2.2.1, 22.2.2 and 2.2.2.3. The coumarins used as reference compounds include: aesculetin, scopoletin, isoscopoletin, scoparone, fraxetin, fraxidin and isofraxidin . All of the coumarins except isofraxidin were obtained from Aapin Chemical Co. Ltd., Oxon. U.K. Isofraxidin was obtained from Yang Shi-lin of the Department of Pharmacognosy, The School of Pharmacy; and it was identified by spectroscopic techniques before use.

97 Methods used for the identification of secondary metabolites in extracts of A. altissima cultures include TLC and HPLC. For the quantification of secondary metabolites in extracts of A. altissima HPLC was employed.

2.2.2.1 Identification of Alkaloids and Coumarins in A. altissima Cell Cultures by Thin Layer Chromatography (TLC) Thin layer chromatography of the analytical type was used for the identification of secondary metabolites in extracts of A. altissima cultures, as well as to establish the purity of reference compounds. TLC was carried out on silica gel 60 F-254 plates (Merck, TLC precoated Aluminium Sheets, Silica gel 60 F 254, E. Merck, Ltd., Darmstadt), to which 1 cm bands of the extracts or compounds to be analyzed were applied. The plates were then developed chromatographically in a suitable solvent system and subsequently visualised under ultraviolet (UV) light at 254 and 366 nm. The canthin- 6-one alkaloids and coumarins were identified by comparing their Rf values and absorbance in UV light at 254 and 366 nm, with those of standard canthin- 6-ones and coumarins. The Rf values of standard canthin- 6-one alkaloids and coumarins, presented in Tables 2-12 and 2-13 respectively (Sub-Section 2.4.1), were determined using the solvents systems given below. The Rf values of these compounds as well as their absorbance in UV light at 254 and 366 nm, were used for their identification by comparing them with those of standard canthin- 6-one alkaloids and coumarins.

2.2.2.1.1 Solvent Systems used for TLC of Alkaloid Reference Compounds The solvent systems used for the TLC of the reference canthin- 6-one alkaloids, are given below. These solvent systems were also used for the separation and identification of the canthin- 6-one alkaloids in extracts of A. altissima cell cultures. 1. Ethyl acetate 2. Dichloromethane : Methanol 80 : 20 3. Chloroform : Methanol 80 : 20 4. Chloroform : Methanol 95 : 5

98 2 .2 .2 .1 . 2 Solvent system used for the TLC of coumarin Reference compounds The solvent system used for the TLC of the coumarin reference compounds is given below. This solvent system was also used for the separation and identification of coumarins in A. altissima extracts. 1. Ethyl acetate

2.2.2.2 Identification and Quantification of Alkaloids and Coumarins of A. altissima by High performance Liquid Chromatography (HPLC) Reversed phase HPLC was used for the identification and quantification of the alkaloid and coumarin constituents of A. altissima.

2.2.2.2.1 Identification of Alkaloids of A. altissima by HPLC The canthin- 6-one alkaloid constituents of A. altissima were identified by HPLC analysis on an Altex isocratic liquid chromatograph (Model 330) with an analytical Hichrom Spherisorb S50DS column, (5pM, C-18) of length: 25 cm and internal diameter: 0.5 cm. The mobile phases used for the separation and subsequent identification of the canthin- 6-one alkaloids include 55 % methanol, which was used for 1-hydroxycanthin- 6-one and 65 % methanol, for canthin- 6-one and 1- methoxycanthin- 6-one. The methanol used was of HPLC grade and had been filtered through a Millepore filter: pore size 0.45 pm in a Millepore filtration system, and degassed by vacuum, prior to use. The column was run at a flow rate of 1 ml . minute '. Detection of the canthin- 6-one alkaloid constituents of A. altissima was by their UV absorption at 280 nm, using an Altex 330 UV detector and Kratos GM-970 fluorescence detector, with emission and excitation wavelengths set at 438 and 403 nm respectively. Both detectors were linked in series to a Kipp and Zonen BD-41 chart recorder. The UV detector was used for the detection of canthin- 6-one and 1- methoxycanthin- 6-one which absorb at 280 nm, whilst the fluorometer was used for the detection of 1 -hydroxycanthin- 6-one which fluoresces at the previously stated emission and excitation wavelengths.

99 The retention times of these alkaloids (stated in Table 2-14, Sub-Section 2.4.1), were used for their identification.

2.2.2.2.2 Quantification of Alkaloids ofA. altissima by HPLC The alkaloids present in extracts of A. altissima cell cultures were quantified using the HPLC column described for their identification, and it was run under conditions similar to those previously described for the identification of alkaloids. The column was calibrated for each of the canthin- 6-one alkaloids (canthin- 6-one, 1 -hydroxycanthin- 6-one and 1 -methoxycanthin- 6-one). Calibration of the HPLC column for the canthin- 6-one alkaloids, was carried out as follows: For 1 -hydroxycanthin- 6-one standard solutions of 1 -hydroxycanthin- 6-one of concentrations ranging from 3.125 to 25 pg . ml * were employed for column calibration. Each of the standard solutions of l-hydroxycanthin- 6-one was prepared in HPLC grade methanol. 20 pi aliquots of each of these standard solutions were then injected on the column and the area under the peak determined by a previously programmed integrator. Duplicate determinations were done at each concentration. A linear calibration curve of peak area versus concentration of 1- hydroxycanthin- 6-one was then plotted. This was subsequently used for the determination of concentrations of l-hydroxycanthin- 6-one present in aliquots of unknown samples. Calibration of the column for canthin- 6-one and l-methoxycanthin- 6-one were achieved by using the procedure previously described for l-hydroxycanthin- 6-one. Concentrations of standards used for calibrating the column for canthin- 6-one and 1-methoxycanthin- 6-one are 0.5 to 2 mg . ml ' and 1 to 4 mg . ml * respectively.

2.2.2.2.3 Identification of Coumarins of A. altissima by HPLC The identifiable coumarins present in A. altissima suspension cultures are scopoletin and isofraxidin . Identification of these coumarins was by using an isocratic Altex liquid chromatograph, with an analytical Hichrom Spherisorb S50DS

100 column (5 pm, C-18), of height: 25 cm and internal diameter: 0.5 cm, linked to it. The mobile phase was 40 % methanol and constituents of extracts of ^4. altissima were detected by their UV absorption at 280 nm. The retention times of scopoletin, and isofraxidin were used for their identification by comparing them with the retention times of standard coumarins (stated in Table 2-15, Sub-Section 2.4.1).

2.2.2.2.4 Quantification of Coumarins of A. altissima by HPLC Quantification of levels of scopoletin and isofraxidin were achieved by using the HPLC column and mobile phase, described for their identification. The column was calibrated for each of these coumarins by using the method described for the canthin- 6-one alkaloids in Sub-Section 222.1.1. Concentrations of standard solutions used were 3.125 to 12.5 pg . ml^ for the calibration of this column for both scopoletin and isofraxidin . A linear calibration curve of peak area versus concentration of each coumarin i.e. scopoletin and isofraxidin was then plotted. These curves were used for the determination of concentrations of scopoletin and isofraxidin present in aliquots of unknown samples.

2.2.2.3 Spectroscopic Methods for the Qualitative Identification of Secondary Metabolites of A. altissima The identity of the canthin- 6-one alkaloid and the coumarin standards and those present in A. altissima cell cultures, were confirmed by employing methods which include the following: fluorescence spectroscopy, ultraviolet spectroscopy (UV), mass spectroscopy (ms) and nuclear magnetic resonance spectroscopy (nmr). The fluorescent, UV, ms and nmr spectra of the canthin- 6-one alkaloids and coumarins of A. altissima are presented in Sub-Section 2.4.2.

2.2.2.3.I Fluorescence Spectroscopy The fact that 1 -hydroxycanthin- 6-one is highly fluorescent made detection by

101 fluorescence possible, l-hydroxycanthin- 6-one has a bright purple fluorescence which is easily visible when dissolved in methanol. The fluorescent spectrum of 1 -hydroxycanthin- 6-one was determined on a Kratos fluorescence spectrophotometer. The spectra were recorded in spectroscopic grade methanol, and the emission and excitation wavelengths recorded. Detection of 1 -hydroxycanthin- 6-one during HPLC analyses was achieved by linking the fluorescent spectrophotometer set at the predetermined emission and excitation wavelengths to the HPLC system described previously in Sub-Section 2.2.22.

2.2.2.3.2 Ultraviolet Spectroscopy The UV spectra of the canthin- 6-one alkaloids and coumarins used as reference compounds were obtained with a Perkin-Elmer 402 UV-visible spectrophotometer. The spectra were recorded in spectroscopic grade methanol.

2.2.2.3.3 Mass Spectroscopy Electron impact Mass spectra of the canthin- 6-one alkaloid and coumarin reference compounds, were recorded on a VG Analytical LTD ZAB IF Spectrophotometer at 70 eV. The determinations were made in spectroscopic grade methanol or chloroform.

2.2.2.3.4 Proton-Nuclear Magnetic Resonance Spectroscopy Spectra were obtained on a Bruker WP, 60 MHz, a Bruker WM, 250 MHz or on a Bruker WS, 400 MHz Spectrophotometer. Chemical shifts were recorded on the 5-scale relative to tetramethylsilane. The determinations were made in spectroscopic grade deuterated chloroform or methanol.

102 2.3 Methods used for the Extraction, Isolation and Purification of Methyltransferases from A. altissima

2.3.1 Preparation of Buffers The procedures used by Dawson et a l (1986 a) were adapted for the preparation of the required buffers.

2.3.1.1 Homogenisation Buffer (A) The homogenisation buffer (A), which was used for enzyme extraction from harvested cell cultures of A. altissima, is 100 mM sodium phosphate buffer pH 7.5 containing 60 mM p-mercaptoethanol (p-MCE) (Sigma Chemical Company Ltd, U.K.). 87 ml of 200 mM di-sodium hydrogen phosphate, Na 2HP04 (British Drug Houses Poole, Dorset) and 13 ml of 200 mM sodium di-hydrogen phosphate, NaH 2P04 (British Drug Houses, Poole, Dorset), were thoroughly mixed with 100 ml of distilled water added to obtain 200 ml of a 100 mM solution of sodium phosphate buffer. A pH determination was done with a Phillips PW 9418 pH meter to ensure that the pH of the solution was at the required level of 7.5. A volume of p-MCE required to give 60 mM levels in the homogenisation buffer, was then added to the solution.

2.3.1.2 Running Buffer (B) Running buffers refer to the buffers used for the equilibration and elution of proteins from the various columns employed during the enzyme purification procedure. Running Buffer B(I) This constitutes 100 mM sodium phosphate buffer pH 7.5, containing 20 mM p-MCE. It was prepared as described for the homogenisation buffer (A), but running buffer B(l) contained only 20 mM levels of p-MCE. Running buffers used on pre-packed Pharmacia columns i.e. (the Pharmacia Mono-Q HR 5/5 and the Pharmacia Superose 12 HR 12/30 columns), were prepared with Milli-Q water instead of distilled water and were subsequently filtered through a Millepore filter (pore size 0.45 pm), using a Millepore filtration system.

103 Running Buffers B(2) These refer to the buffers used for running potassium chloride (KCl) gradients on the ion exchange columns employed during the enzyme purification procedure. These are basically Running buffer B(l) containing varying molar concentrations of KCl. Running Buffer B(2)-l Running buffer B(2)-l is 100 mM sodium phosphate buffer, pH 7.5, which contains 50 mM levels of potassium chloride and 20 mM p-MCE. Running buffer B(l) was prepared as previously described and KCl added to obtain 50 mM levels in running buffer B(l). Running Buffer B(2)-2 Running buffer B(2)-2 is 100 mM sodium phosphate buffer, pH 7.5, which contains 200 mM levels of KCl and 20 mM p-mercaptoethanol. Running buffer B(l) was prepared and KCl added to obtain 200 mM levels of KCl in running buffer B(l). Running Buffer B(2)-3 Running buffer B(2)-3 is 100 mM sodium phosphate buffer, pH 7.5, which contains 1 M levels of KCl and 20 mM p-MCE. This was prepared as previously described for B(2)-l and B(2)-2, but contained 1 M KCl levels. Milli-Q water was used for the preparation of this buffer. The buffer was subsequently filtered through a Millepore filter (pore size 0.45 pm), using a Millepore filtration system. Running Buffers B(3) These refer to buffers used for running a phosphate gradient on the hydroxylapatite columns employed during the enzyme purification procedure. They basically constitute running buffers, which contain varying molar levels of sodium phosphate at pH 7.5, as well as 20 mM P-MCE. These buffers include the following: B(3)-l: 20 mM sodium phosphate buffer pH 7.5 + 20 mM p-MCE B(3)-2: 80 mM sodium phosphate buffer pH 7.5 + 20 mM P-MCE B(3)-3: 140 mM sodium phosphate buffer pH 7.5 + 20 mM P-MCE

104 20 mM, sodium phosphate buffer was obtained from solutions of di-sodium hydrogen phosphate (NazHPO^) and sodium di-hydrogen phosphate (NaH 2? 04) of a corresponding molarity. These solutions were combined as follows for the preparation of 20 mM sodium phosphate buffer: 87 ml of Na 2HP04 and 13 ml of NaH 2P04 were measured and NaH 2P04 was slowly added to Na 2HP04, and mixed thoroughly whilst checking the pH concurrently. When the required pH of 7.5 was attained, addition of NaH2P04 was halted and the solution finally made up to 100 ml with distilled water. The pH was rechecked at this point to ensure that it remained at the required level. The above procedure was repeated for the preparation of 80 mM and 140 mM sodium phosphate buffers, from solutions of Na 2HP04 and NaH 2P04 of concentrations of 80 and 140 mM respectively.

2.3.1.3 Buffers for the Determination of Optimum pH (C) These buffers were used for the determination of optimum pH for enzyme activity. They were used for adjusting the pH of the partially purified A. altissima methyltransferase enzyme solutions to the required levels during optimum pH determination. The pH levels investigated are: 5.6, 6, 6.6, 7, 7.5, 8 , 8.5 and 9. Addition of 50 pi of buffer of the required pH to 50 pi of the partially purified enzyme solution, was sufficient to adjust the pH of the enzyme solution to the required level, prior to carrying out enzyme assays. Buffers C(l) Running buffers C(l) refer to, 400 mM citric acid : sodium citrate buffers of varying pH levels. These buffers which also contain 20 mM p-MCE, include the following: C(l)-1: 400 mM citric acid : sodium citrate buffer pH 5.6 C(2)-2: 400 mM citric acid : sodium citrate buffer pH 6.0

100 ml of each of these buffers was prepared from predetermined volumes of 400 mM citric acid and 400 mM sodium citrate (Dawson et ah, 1986 a). The citric acid was slowly added to the sodium citrate, whilst checking the pH concurrently.

105 When the required pH was attained, addition of citric acid was halted and the solution made up to volume with distilled water. Buffers C(2) Running buffers C(2) refer to 400 mM sodium phosphate buffer of varying pH levels. These buffers which also contain 20 mM |3-MCE include the following: C(2)-l: 400 mM sodium phosphate buffer pH 6.0 C(2)-2: 400 mM sodium phosphate buffer pH 6.6 C(2)-3: 400 mM sodium phosphate buffer pH 7.0 C(2)-4: 400 mM sodium phosphate buffer pH 7.5 C(2)-5: 400 mM sodium phosphate buffer pH 8.0

100 ml of each of these buffers was prepared from pre determined volumes of 400 mM NaH2?04 and 400 mM Na 2HP04 (Dawson et al., 1986 a). The NaH 2P04 was slowly added to the Na 2HP04 whilst checking the pH concurrently. When the required pH was attained, addition of 400 mM NaH 2P04 was halted and the solution made up to volume with distilled water. The pH was then rechecked to ensure that it remained at the desired level. Buffers C(3) These constitute 400 mM Tris hydroxymethylaminomethane-Hydrochloric acid (Tris- HCl) buffer of varying pH levels, with each of them containing 20 mM p-MCE. These buffers include the following: C(3)-l: 400 mM Tris-HCl buffer pH 8.0 C(3)-2: 400 mM Tris-HCl buffer pH 8.5 C(3)-3: 400 mM Tris-HCl buffer pH 9.0

1(X) ml of each of these buffers was prepared from pre-determined volumes of 400 mM Tris and 0.2 N HCl (Dawson et ai, 1986 a). The 0.2 N HCl was slowly added to the Tris, whilst checking the pH concurrently. When the required pH was attained, addition of HCl was halted and the solution made up to volume with distilled water. The pH was rechecked to ensure that it was still at the desired level.

106 2.3.2 Enzyme Extraction from A. altissima Cell Suspension Cultures A. altissima cell suspension cultures were harvested between days 14 to 22 of the growth cycle depending on the cell line used. 40 g of cells were frozen rapidly under liquid nitrogen and allowed to thaw in a glass mortar in 30 to 40 ml of cold homogenisation buffer A. Acid washed sand was then added to the cells, which were thoroughly homogenised with a pestle. The slurry was filtered through a nylon bolting cloth and the filtrate stored in an ice bath, whilst the residue was transferred back into the mortar for a repetition of the homogenisation and subsequent filtration procedures. The filtrates were then bulked and centrifuged for 15 minutes at 10,000 rpm in a refrigerated centrifuge (MSE, High Speed 18). The cell debris settled at the bottom of the tube and the supernatant was decanted and stored in an ice bath after its volume had been determined. The supernatant is described as the crude dialysate.

2.3.3 Assay for Canthin-6 -one Alkaloid and Coumarin Methyltransferase Activities in Cell Suspension Cultures of A. altissima The methyltransferase enzyme was assayed for catalytic activity by monitoring the transfer of the '^CHg label from "‘CHj-S-adenosyl-L-methionine (SAM) to the hydroxyl group of the hydroxylated canthin- 6-one alkaloids and coumarins. The reactions involved are represented as shown in Equation 3.1-100 of Sub-Section 3.1.6.1, for alkaloid méthylation and Equations 3.1-101 and 3.1-102 of Sub-Section 3.1.6.2, for coumarin méthylation. Assays for enzyme activity were carried out in an incubation mix which contained: 1. An enzyme solution; 2. An hydroxylated substrate; 3. Another substrate i.e. '"^C-SAM (S-Adenosyl L-[Methyl *'^C]-methionine solution in sulphuric acid (H2SO4), from Amersham International PLC, Buckinghamshire, England) and sometimes non-labelled SAM (S-Adenosyl L-Methionine Iodide Salt, from Sigma Chemical Company Poole, U.K.). Substrates Used for Methyltransferase Enzyme Assays The hydroxylated canthin- 6-one alkaloids used as substrates in the assay for methyl transferase enzyme activity in A. altissima include: 1 -hydroxycanthin- 6-one and

107 lO-hydroxycanthin- 6-one. lO-hydroxycanthin- 6-one was obtained from Yang Shi-lin of the department of Pharmacognosy, The School of Pharmacy and its identity was subsequently verified by Spectroscopic techniques. The hydroxylated coumarins used as substrates include: aesculetin; scopoletin and isoscopoletin for the investigation of sequence ( 1) of coumarin méthylation, as well as fraxetin; isofraxidin and fraxidin for the investigation of sequence ( 2) of coumarin méthylation. Details of these pathways are shown in Sub-Sections 3.1.6.2 and 3.5.2. The other substrate used is SAM both in the ^'^C-labelled and non-labelled form.

2.3.3.1 Preparation of the Incubation Mix and its Constituents Three types of incubation mixes were used. These include: 1. The Routine Incubation Mix Routine assays for enzyme activity, were carried out on routine incubation mixes to determine the activity of 1-hydroxycanthin- 6-one methyltransferase (1-HMT) enzyme and the coumarin methyltransferase (GMT) enzyme. These assays were used for monitoring protein fractions obtained from columns employed during enzyme purification procedures. Routine assays were also used in certain preliminary and routine experiments. These incubation mixes contained less than saturating levels of the substrates. The constituents of a typical routine incubation mix in a total volume of 130 pi, are shown in Table 2-10.

2. The Standard Incubation Mix Standard assays for enzyme activity, were carried out on standard incubation mixes. These assays were used for experiments done to characterise the 1-HMT enzyme. Standard incubation mixes contained saturating levels of the substrates and assays were carried out under optimum conditions for enzyme activity. The constituents of a typical standard incubation mix in a total volume of 130 pi, are shown in Table 2-11.

108 TABLE 2-10 Constituents of Incubation Mixes Used for Routine Assays in a Total Volume of 130 pi

Constituent Volume Final Concentration in (Ml) the Incubation Mix Protein 100 12 to 600 pg, depending on the level of purification of the enzyme solution Hydroxlated 10 70 pM for Canthin- 6-one Substrate substrates and Coumarin substrates ‘^C-SAM 20 10 pM Running Buffer B(l) To 130 pi, where necessary

Table 2-11 Constituents of Incubation Mixes Used for Standard Assays in a Total Volume of 130 pi

Constituent Volume Final Concentration in (Ml) the Incubation Mix Protein 100 6 to 49 pg, depending on the level of purification of the enzyme solution Hydroxlated 10 230 pM Substrate '^C-SAM + SAM 20 60 pM Running Buffer B(l) To 130 pi, where necessary

109 3. The Control Incubation Mix Two types of control incubation mixes were used: a. The 0 % Control Incubation Mix The enzyme activity corresponding to the 0 % control was obtained by the determination of the enzyme activity in the 0 % incubation mix. The 0 % control was used for the determination of levels of background radioactivity in dpm during an enzyme assay. Enzyme activity in the 0 % control incubation mix is equivalent to the background radioactivity. The 0 % control incubation mix is similar in constitution to the typical routine or standard incubation mixes (shown in Tables 2-10 and 2-11 respectively), but the hydroxylated substrate was excluded. b. The 100 % Control Incubation Mix The 100 % control was used in inhibitor studies to determine the maximum enzyme activity in the absence of inhibitors. This control served as a yardstick for measuring levels of inhibition attained during inhibitor studies.

2.3.3.1.1 Preparation of Hydroxylated Substrates The hydroxylated canthin- 6-one and coumarin substrates used for the methyltransferase enzyme assays are listed under Sub-Section 2.3.3. Stock solutions of these substrates, were made up in a solution containing one part of HPLC grade methanol and three parts of running buffer B(l). The concentrations of each of these stock solutions were such that when 10 pi of a stock solution was pipetted into the incubation mix, it produced the desired substrate concentration in this mix.

2.3.3.1.2 Preparation of SAM Other substrates employed in the assay of the methyltransferase enzyme of A. altissima include radiolabelled SAM (hot SAM) and non-labelled SAM (Cold SAM). Stock solutions of ‘"‘C-SAM of the desired concentrations were made up in running buffer B(l) so that the desired concentration of SAM in the mix, could be

110 produced easily.

Preparation of Hot SAM and Cold SAM Mixtures of radiolabelled SAM (hot SAM) and unlabelled SAM (cold SAM), were used for enzyme assays carried out during the characterisation of the enzyme. In such experiments saturating levels of SAM were required in the incubation mixes. Mixtures of hot and cold SAM were used in order to attain these saturating levels without using excessive levels of hot SAM. Stock solutions of hot SAM and purified cold SAM were prepared in running buffer B(l), so that when calculated volumes of the stock solutions were pipetted into the incubation mix, they produced the desired concentration of SAM. Purification of Cold SAM The purification of cold SAM was carried out to ensure that it was completely free of S-adenosylhomocysteine (SAH) which is known to inhibit SAM competitively. The procedure used for the separation of SAM and SAH is dependent on their differences in binding properties to the anionic resin, Dowex bicarbonate (HCO3 ) at a pH of 7. At this pH, SAH is bound firmly to the Dowex HCO 3, whilst SAM does not bind to this resin. This purification method is a modification of the method used by Shapiro and Ehninger (1966). Dowex HCO 3, was prepared from Dowex Cl resin (Fluka, Dowex 1). The procedure involved soaking 10 g of the Dowex Cl resin overnight in 10 M sodium hydroxide solution followed by vacuum filtration in a Büchner funnel and washing with distilled water until the waste water had a neutral pH. The resin was then washed with 150 ml of 0.5 M sodium bicarbonate solution, followed by 150 ml of water and the pH of the waste water was re-checked to ensure neutrality. The resin was subsequently air dried and packed in a column of dimensions 2.5 cm in height and 1 cm internal diameter. 10 mg of cold SAM in 500 pi of distilled water was applied to the column and the column eluted with 3 ml of distilled water. This was sufficient to elute the SAM, while the SAH was tightly bound to the column and was only eluted from the column with 30 ml of 1 M sodium chloride (NaCl). The fractions containing SAM were bulked and freeze dried. The

111 UV absorbance of SAM was determined before and after freeze drying for identification purposes.

2.3.3.2 Preparation of Other Additives used in the Enzyme Assay

2.3.3.2.1 Preparation of 1 M KCl in 2 % HCl This solution was used, to stop enzyme activity at the end of the incubation period. A stock solution of 1 M KCl in 2 % HCl (1 litre), was prepared by dissolving 74.56 g of KCl in a previously prepared solution of 2 % HCl in distilled water. The solution was then made up to volume and stored in the refrigerator for subsequent use.

2.3.3.2.2 Preparation of Méthylation Products of the Enzyme Reaction 1 -Methoxycanthin- 6-one is the méthylation product of a reaction catalysed by the 1-HMT enzyme of A. altissima (the méthylation of 1-hydroxycanthin- 6-one). 10- methoxycanthin- 6-one is the product of the méthylation of lO-hydroxycanthin- 6-one by the canthin- 6-one methyltransferase enzyme. Possible products of the méthylation reaction catalysed by the CMT enzyme of A. altissima are scopoletin, and isoscopoletin, formed from aesculetin as well as fraxidin and isofraxidin , formed from fraxetin. Aliquots of solutions made from each of these products, were used for spiking each of their corresponding incubation mixes during enzyme assay (as described in Sub-Section 2.3.3.3). Stock solutions of these compounds used as spikes were made up in HPLC grade methanol. Concentrations of these stock solutions were such that when 10 pi of a stock solution was pipetted into the incubation mix, it produced 2 mM levels of the méthylation product. Méthylation products SAH and 1-methoxycanthin- 6-one were also tested for their inhibitory effect on the 1-HMT enzyme. Stock solutions of SAH and 1- methoxycanthin- 6-one, for inhibitory tests were made up in a solution of 50 % HPLC grade methanol and distilled water.

112 2.3.3.2.3 Preparation of Divalent Cations The divalent cations tested for their effect on 1-HMT activity include magnesium, manganese and copper as their sulphate or chloride salts. Stock solutions of these salts obtained from BDH Chemicals Poole, Dorset. U.k. were prepared in a solution of 50 % methanol in distilled water.

2.3.3.2.4 Preparation of Inhibitors Inhibitors investigated for their effect on 1-HMT activity include potassium cyanide, iodoacetamide, N-ethylmaleimide and p-chloromercuricbenzoate, which were obtained from BDH Chemicals, Poole, Dorset. U.k. Stock solutions of these inhibitors were prepared in a solution of 50 % ethanol and distilled water.

2.3.3.3 Enzyme Assays The Assay for methyltransferase activity involved, preparation of the appropriate assay mix of pH 7.5 (i.e. the pH of the enzyme solution) and subsequent incubation at 35 °C for the routine assay and 30 °C for the standard assay, in a water bath (Grant instruments, Cambridge England). An incubation time of 30 or 45 minutes was used for standard assays and 60 minutes for routine assays. The reaction was terminated by the addition of 400 pi of a solution of 1 M KCl in 2 % HCl and 800 pi of toluene was added for product extraction. The incubation mix was then spiked with l-methoxycanthin- 6-one or the corresponding methylated product at 2 mM levels. The presence of higher levels of the methylated product in solution was expected to aid the extraction of the methylated product and also allow for easy identification during TLC, by making the band more visible. The methylated products were extracted by mixing vigorously in a vortex mixer (Hati Rotamixer) for 2 minutes. This turbid mixture was separated by centrifugation in an Eppendorf centrifuge for 5 minutes at 3000 rpm. 600 pi of the organic phase (toluene) was transferred into a scintillation vial, 4.5 ml of scintillant (Scintran Cocktail-T, obtained from British Drug Houses, Poole, Dorset) added, and the sample counted for radioactivity in a Beckman LS-600TA scintillation counter.

113 The enzyme activity (in dpm) is an expression of the amount of product formed during the reaction. Enzyme activity in a routine or standard incubation mix was determined by carrying out enzyme assays on the appropriate incubation mix and the corresponding 0 % control (in dpm). These incubation mixes were prepared as previously described in Sub-Section 2.3.3.1. The accurate enzyme activity of an incubation mix (in dpm) was obtained by subtracting the background radioactivity i.e. (the activity corresponding to the 0 % control), from the actual enzyme activity obtained during the assay.

2.3.3.4 Calculation of Enzyme Activity in Pkat A Pkat of enzyme activity is defined as the conversion of 1 picomole of substrate per second. Enzyme activity in PKat, was determined by using Equation 2- 100, which is a modification of the equation used by Wolf et al. (1983). Equation 2- 100 was used for the conversion of radioactive counts for enzyme activity in dpm to Pkat.

Equation 2-100

R n V Z = Pkat 2.22 X 10'^ X SA X 10 " x t x V, where; Z = Enzyme activity in Pkat.

Rn = Net counting rate in disintegrations per minute. V = Volume used for product extraction in litres. 2.22 X 10*^ = Factor for conversion of decay rate in dpm to curie (Q). SA = Specific radioactivity in Q per mole of substrate. 10 = Factor for conversion of mole to picomole. t = Time in seconds.

114 V, = Volume removed for scintillation counting in litres.

2.3.3.S Product Identification Experiments were carried out in order to positively identify the products of the 1 -hydroxycanthin- 6-one alkaloid and coumarin méthylation reactions catalysed by the methyltransferase enzymes of A. altissima. The méthylation products of l-hydroxycanthin- 6-one and lO-hydroxycanthin- 6- one are 1-methoxycanthin- 6-one and lO-methoxycanthin- 6-one respectively. Two sequences for coumarin méthylation were investigated i.e. sequence 1 for the monomethylation of aesculetin to scopoletin and or isoscopoletin and sequence 2 for the monomethylation of fraxetin to fraxidin and or isofraxidin (shown in Sub- Sections 3.1.6.2 and 3.5.2). Routine enzyme assays were carried out on incubation mixes containing the relevant hydroxylated substrate e.g. l-hydroxycanthin- 6-one, aesculetin or fraxetin. Enzyme assays and subsequent extractions of products were carried out as described in Sub-Section 2.3.3.3. The compounds listed below were individually spotted in 1 cm bands on a pre-coated silica gel 60F-254 plate. These include: The extracted product from each incubation mix assayed; a standard solution of the relevant méthylation product e.g. l-methoxycanthin- 6-one for 1-HMT assay and scopoletin or isofraxidin for the CMT assay; a standard solution of the relevant hydroxylated substrate e.g. 1 -hydroxycanthin- 6-one for the 1-HMT assay and aesculetin or fraxetin for the CMT assay. The silica gel plate was then developed in a solvent system of dichloromethane : methanol 80 : 20 or ethyl acetate for the 1- HMT assay and ethyl acetate for the coumarin methyltransferase assay. The TLC chromatogram was divided into 3 columns. The column of the chromatogram containing the methylated product was further divided into 1 cm bands whilst monitoring under UV light at 254 nm, to ensure that the developed spots were fully contained within a band. The chromatogram was then traced and the column containing the methylated product was subsequently cut up into 1 cm bands. Each

115 disc was placed in a separate scintillation vial and 300 pi of HPLC grade methanol added to each vial and allowed to stand for 1 hour with intermittent gentle swirling, to allow for the elution of the methylated product into the solvent. 4.5 ml of scintillant was then added to each vial and the radioactivity in each band determined in disintegrations per minute.

2.3.3.6 Protein Determination Protein levels in the various enzyme preparations were determined by employing the method of Bradford (Bradford, 1976).

2.3.3.6.1 Preparation of Protein Reagent (Bradford’s Reagent) 100 mg of coumassie brilliant blue G-250 (Sigma Chemical Company Ltd., U.K.), was dissolved in 50 ml of 95 % ethanol. To this solution, 100 ml of 85 % w/v phosphoric acid was added and the resulting solution diluted to a final volume of 1 litre. This solution was then filtered to remove sediments. Final concentrations in the reagent were 0.01 % w/v coumassie brilliant blue G-250, 4.75 % v/v ethanol and 8.5 % v/v phosphoric acid.

2.3.3.6.2 Protein Assay A stock solution containing 1 mg . ml ' of BSA protein, (obtained from Sigma Chemical Company Ltd., U.K.), was prepared. Volumes of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 pi, corresponding to similar pg levels of protein, were individually pipetted into separate test tubes and 5 ml of Bradford’s reagent added. The contents were immediately mixed by inversion and the absorbance of the solution determined at 595 nm in a UV-Visible spectrophotometer (Pye Unicam SP8-100 UV spectrophotometer), between 2 minutes and 1 hour after preparation. The determinations were done in 3 ml cuvettes against a blank control (Bradford’s reagent without added protein). A calibration curve of protein concentration versus corresponding absorbance was plotted and protein levels of unknown samples ( 20, 50 or 100 pi of protein in 5 ml of Bradford’s reagent), were determined by interpolation from the calibration curve.

116 2.3.4 Purification of Methyltransferases from A. altissima Suspension Cultures The crude dialysate obtained from A. altissima cell suspension cultures, which was prepared as described in Sub-Section 2.3.2 was further purified by employing the following methods.

2.3.4.1 Removal of Aromatic Compounds with XAD-2 Resin 0.1 mg . ml ' of Amberlite XAD-2 resin (British Drug Houses, Poole, U.K), was added to the crude dialysate (prepared as previously described in Sub-Section 2.3.2). The solution was swirled gently and then allowed to stand for 15 minutes, after which the XAD-2 resin was removed by filtration, through cotton wool which was washed with methanol and dried prior to use.

2.3 4.2 Ammonium Sulphate [(NHJgSOJ Precipitation of Proteins An ammonium sulphate precipitation of proteins from the enzyme solution was done, in order to obtain a precipitate at 0 to 40 % (NH 4)2S04 saturation and subsequently a second precipitate at 40 to 70 % saturation. With the aid of an ammonium sulphate precipitation chart (Dawson et al., 1986 b), it was possible to determine the quantities of ammonium sulphate required to achieve these saturation levels. To attain 0 to 40 % saturation, 22.9 g of (NH 4)2S04 for enzyme work ((NH4)2S04, specially low in heavy metals, obtained from British Drug Houses, Poole. U.K), per 100 ml of enzyme solution was used. The required amount of (NH 4)2S04 was weighed and finely ground in a glass mortar. This was gradually dissolved in the enzyme solution by gently stirring it in, with a glass rod. The solution was then allowed to stand in an ice bath for 60 minutes after which it was subsequently centrifuged for 15 minutes at 10,000 rpm in the refrigerated centrifuge. The supernatant was then decanted into a beaker and the 0 to 40 % precipitate stored in the ice bath. In order to obtain 40 % - 70 % (NH 4)2S04 saturation, an additional 19 g of finely ground (NH 4)2S04 per 100 ml of enzyme solution was slowly dissolved in the supernatant and the precipitation procedure repeated. The resulting 40 to 70 % precipitate was then stored in the ice bath for further purification.

117 2.3.5 Further Purification of the 40 % - 70 % (NHJ2 SO4 Protein Precipitate The 40 % - 70 % Ammonium sulphate protein precipitate obtained as described in Sub-Section 2.3.4.2, was further purified using a combination of 3 or more of the following purification methods, though not necessarily in the order presented. The combination of methods and the order in which they were employed which are termed "purification sequences" are described in detail in Section 3.3.

2.3.5.1 Method 1: Desalting the 40 % - 70 % (NHJ2 SO4 Protein Precipitate The precipitate was reconstituted in 5 ml of running buffer B(l) and was divided into two batches of 2.5 ml volume, which were applied to Pharmacia PD-10 columns (containing coarse sephadex G.25). The columns had been pre-equilibrated with running buffer B(l). The phenolic materials as well as the (NH 4)2S04 salt, were absorbed onto the column whilst the relevant proteins were not. These proteins were then eluted from the column with 3.5 ml of running buffer B(l), and bulked.

2.3.5.2 Method 2: Diethylaminoethyl (DEAË) Cellulose Anion Exchange Chromatography A column of dimensions, 3.5 cm in height and 1.7 cm internal diameter was packed with microgranular DEAE cellulose (DEAE Cellulose D-52, Whatman Ltd. Maidstone, England), and then equilibrated with running buffer B(l). 6 ml of desalted protein solution was then applied to the column and the protein eluted with a stepwise KCl gradient of 0 to 200 mM KCl. The elution profile used is as follows: 15 ml of running buffer B(l); 8 ml of running buffer B(2)-l (running buffer B(l) + 50 mM KCl) and 12 ml of running buffer B(2)-2 (running buffer B(l) + 2(X)mM KCl). 2 ml fractions were collected at a flow rate of 0.7 ml . min ', with an automated fraction collector (LKB Ultrorac Fraction Collector- 7000, LKB, Sweden). The proteins eluted from the column were detected, by a UV detector, (LKB UV CORD-2 detector unit, type 8303 A, LKB, Sweden), which was set at 280 nm, and

118 linked to a chart recorder (Type BD-40 Kipp and Zonen, Delft, Holland). The fractions were then assayed for l-hydroxycanthin-6-one and coumarin methyltransferase activities as described in Sub-Section 2 3 3 3 . This column was found to separate the hydroxycanthin-6-one and coumarin methyltransferase activities. The CMT enzyme was not bound to the column and was therefore eluted with the void volume whilst the 1-HMT enzyme was bound to the column and was only eluted after the application of the KCl gradient. The active fractions for each methyltransferase activity were pooled and stored at 4 °C. The bulked 1-HMT fractions were used for protein determination, application to the next purification step and characterising the enzyme. The bulked CMT enzyme was used for protein determination and characterisation of the enzyme.

2.3 5.3 Method 3: Hydroxylapatite Column Chromatography A column of dimensions 3.5 cm in height and 1.7 cm internal diameter, was packed with hydroxlyapatite (Bio-Gel HTP, from Bio-rad Laboratories, California, U.S.A) and equilibrated with running buffer B(l). 6 ml of the desalted protein solution was then absorbed onto the column and a stepwise elution performed using the following amounts of sodium phosphate running buffers of the B(3) range, which contained different concentrations of sodium phosphate (Sub-Section 2.3.1). The elution profile used is as follows: 14 ml of 20 mM sodium phosphate (Na 2P04) buffer (buffer B(3)-l); 20 ml of 80 mM Na2Po4 buffer (buffer B(3)-2); and 30 ml of 140 mM Na 2P04 buffer (buffer B(3)-3). 2 ml fractions were collected at a flow rate of 0.8 ml . min.'L The proteins eluted from the column were monitored, using the same detection system described for the DEAE cellulose procedure (method 2, Sub-Section 2.3.5.2). The fractions were subsequently assayed for 1-HMT and CMT activities and active fractions for each methyltransferase activity pooled for application to the next purification step and for protein determination.

119 2 3.5.4 Method 4 Sephadex G-50 Gel Filtration Chromatography A column, 30 cm in height and 1.7 cm internal diameter was packed with coarse Sephadex G-50 obtained from Pharmacia. The column was pre-equilibrated with running buffer B(l), and 6 ml of enzyme solution applied to it. The proteins were then eluted with running buffer B(l), at a flow rate of 1 ml . min. ' and 2 ml fractions collected. Detection of proteins and recording were as previously described for method 2 (Sub-Section 2.3.5.2). The fractions were then assayed for 1-HMT and CMT activities and active fractions bulked for use in the next purification step and for protein determination.

2.3.5.5 Method 5: Sephacryl S-200 Gel Filtration Chromatography High resolution Sephacryl S-200 obtained from Pharmacia was packed into a column of 30 cm in height and 1.7 cm internal diameter and pre-equilibrated with the running buffer B(l). 5 ml of enzyme solution, requiring further purification was applied to the column and the proteins eluted with running buffer B(l), at a flow rate of 1 ml . min This flow rate was attained with the aid of a peristaltic pump. 1 ml fractions were collected and the proteins eluted from the column, were monitored using the detection and recording systems described for method 2 (Sub-Section 2.3.5.2). The fractions were then assayed for 1-HMT and CMT activities. The active fractions were bulked for application to the next purification step and protein determination.

2.3 5.6 Method 6: Mono-Q Anion Exchange Chromatography A pre-packed Pharmacia Mono Q HR 5/5 column of dimensions 5 cm in height and 0.5 cm internal diameter was linked to a Pharmacia FPLC system. The FPLC system was also linked to a UV detector (Pharmacia single path monitor UV-1 Pharmacia, Sweden), for detection of proteins at 280 nm and a Pharmacia chart recorder. The column was equilibrated with running buffer B(l) and 10 to 30 ml of the desalted appropriate protein preparation applied to it, by means of a 10 ml Pharmacia superloop. The FPLC system was programmed to run a KCl gradient of

120 0 to 0.5 M KCl over 20 minutes, followed by another gradient of 0.5 to 1 M KCl over 5 minutes, at a flow rate of 1 ml . min. 0.5 or 1 ml fractions were collected using an automated fraction collector (Pharmacia, Frac-100). The fractions were subsequently assayed for 1-HMT activity and active fractions pooled, for the purpose of protein determination, further purification and characterisation of the enzyme.

2.3.5.7 Method 7: Q-Sepharose Fast Flow Anion Exchange Chromatography A column, 2 cm in height and 1 cm internal diameter was packed with Pharmacia Q-Sepharose fast flow resin. This was linked to a Pharmacia FPLC system, which was also linked to a UV detector (Pharmacia single path monitor UV-1), set to 280 nm and a Pharmacia chart recorder. The column was equilibrated with running buffer B(l), and 10 to 30 ml of the desalted appropriate protein preparation applied to it by means of a 10 ml Pharmacia superloop. The FPLC system was programmed to run a KCl gradient of 0 to 1 M KCl over 87 minutes, at a flow rate of 1 ml . min *. 1 ml fractions were collected with an automated Pharmacia fraction collector (Frac- 100). The fractions were subsequently assayed for 1-HMT activity and active fractions pooled for the following purposes; protein determination, further purification and characterisation of the enzyme.

2.3.5.8 Method 8: Gel Filtration on Pharmacia Superose 12 HR 10/30 Column A prepacked Pharmacia Superose 12 HR 10/30 column of 30.5 cm in height and 1.5 cm internal diameter was linked to the Pharmacia FPLC system, UV detector and Chart recorder described in methods 6 and 7 (Sub-Sections 2.3.5.6 and 23.5.1). The column was equilibrated with running buffer B(l) prior to the application of 0.2 to 2 ml of the appropriate enzyme solution by means of a 1 ml syringe and needle. The proteins were eluted from the column with running buffer B(l), at a flow rate of 0.4 ml . min. '. 0.5 or 1 ml fractions were collected in the automated fraction collector described in methods 6 and 7 (Sub-Sections 2.3.5.6 and 2.3.5.V) and the fractions were assayed for 1-HMT activity. The active fractions were pooled for enzyme characterisation.

121 2.3.6 Characterisation of the 1-HMT Enzyme Characterisation of the 1-HMT enzyme involved carrying out the following experiments: determination of optimum conditions for enzyme activity (Sub-Section 3.4.1); determination of kinetic properties of the enzyme substrates (Sub-Section 3.4.2); inhibitor studies (Sub-Section 3.4.3); molecular weight determination (Sub- Section 3.4.4) stability studies (Sub-Section 3.4.5) and substrate specificity studies (Sub-Section 3.4.6). These determinations (with the exception of the molecular weight determinations), were not elaborated upon in this Section because they have been described in detail in their respective Sections.

2.3.6.1 Molecular Weight Determination The molecular weight of 1-HMT was determined, after the final purification step in purification sequence 1 (described in Sub-Section 3.3.1). The determination was done on the active Pharmacia Superose 12 HR 10/30 column eluate from purification sequence 1. Two methods were used for molecular weight determination; a gel filtration method which involved the use of the Pharmacia Superose 12 HR 10/30 column and an SDS-Polyacrylamide gel electrophoresis method.

2.3.6.1.1 Molecular weight Determination by the Gel Filtration Method The molecular weight of the purified enzyme was determined by comparing the elution volume of the enzyme on a Pharmacia Superose 12 HR 10/30 column with the elution volumes of a mixture of molecular weight markers, which had previously been run on the same column, under the same conditions. The molecular weight markers used include aldolase, egg albumin, chymotrypsinogen A and cytochrome C of molecular weights 158,000, 45,000, 25,000 and 12,500 respectively. A mixture of molecular weight markers containing 1 mg . ml ' of each of the markers was made up in running buffer B(l). A 100 pi solution of this mixture was injected on the column and proteins eluted with running buffer B(l) at a flow rate of 0.4 ml . min '. The elution volumes of each of the molecular weight markers was determined and a calibration curve of molecular weight versus the elution volume of each marker was plotted.

122 A 200 \i\ solution of the Mono-Q eluate from purification sequence 1 containing 45 pg of protein was injected on the column and proteins eluted with running buffer B(l) at a flow rate of 0.4 ml . min. 1 ml fractions were collected and assayed for methyltransferase activity. The elution volume of the active fraction was determined and the molecular weight obtained from the calibration curve.

2.3.6.1.2 Molecular weight Determination by Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Vertical SDS-PAGE, was performed simultaneously on a mixture of molecular weight markers, and on samples of the purified 1-HMT enzyme using a 15 % polyacrylamide gel of 1.5 mm thickness. Gel Preparation A 15 % SDS-Polyacrylamide gel was prepared, from a mixture containing: 10 ml of 0.5 M Tris-HCl buffer pH 6.8; 20 ml of a 30 % acrylamide stock solution which contained (29.2 g of ultrograde acrylamide from LKB Sweden + 0.8 g of bis- acrylamide from LKB Sweden per 100 ml of solution); 0.4 ml of a 10 % SDS solution and 9.4 ml of milli-Q water. The mixture was thoroughly degassed and 200 pi of a solution of 10 % Ammonium persulphate and 20 pi of TEMED (ultrograde TEMED, LKB, Sweden), were added to the solution, prior to rapidly pouring it into a cast, where the gel solution was left standing until it set. The gel comb was then placed in the space on top of the cast and a 7.5 % stacking gel was prepared from a solution containing: 5 ml, 1.5 M Tris-HCl buffer, pH 8.8; 10 ml of the 30 % acrylamide stock solution; 0.2 ml of 10 % SDS and 24.6 ml of Milli-Q water. This solution was also thoroughly degassed prior to the addition of 200 pi of a 10 % solution of ammonium persulphate and 20 pi of Temed. The stacking gel solution was then poured into the cast, where it was left standing until it set. The gel comb was subsequently removed to reveal the wells formed for sample application. Sample Treatment by the Laemlli Method The samples to be applied to the gel were treated according to the Laemlli

123 method (Laemlli, 1970). These samples include the purified 1-HMT enzyme solution as well as a mixture of molecular weight markers, containing the following proteins: bovine carbonic anhydrase, hen egg ovalbumin, bovine serum albumin, phosphorylase b, P-galactosidase and myosine of molecular weights 29000, 45000, 66000, 97000, 116000 and 205000 respectively. This standard mixture was obtained from Sigma Chemical company U.K. (MW SDS-2(X) molecular weight Markers). Laemlli sample buffer, which was prepared according to the method of Laemlli (1970), contained: 0.0625 M Tris-HCl pH 6.8; 2 % SDS w/v; 10 % glycerol v/v; 5 % p-MCE v/v; 0.001 % bromophenol blue w/v). A 1 mg . ml ' solution of the molecular weight markers was prepared in Laemlli buffer and the 1-HMT enzyme solution was prepared for SDS-PAGE by mixing 200 pi of the enzyme solution with an equal volume of the Laemlli buffer. These solutions were then immersed in boiling water for 5 minutes, after which 20 to 30 pi of the standard molecular weight markers and 50 to 1(X) pi of the purified enzyme solution, were applied to each well in the gel. The gel was run on a vertical electrophoresis unit using an LKB 2197 Electrofocusing constant power supply (LKB Sweden), under the following conditions: Constant current, | 30 mA; temperature, 14.5 °C and running buffer pH, 8.3; containing 15 g of Tris (Sigma Chemical Co. Ltd U.K.), 72 g of Glycine (Analar Glycine aminoacetic acid, British Drug Houses, Poole, Dorset) and 5 g of SDS (LKB, Sweden) in 5 litres of distilled water. The developed gel was fixed overnight in a fixing solution containing 30 % ethanol and 10 % acetic acid in milli-Q water. Gel Staining The fixed gel was transferred into a coumassie brilliant blue solution (Coomassie Blue G-250, obtained from British Drug Houses, Poole, Dorset) for staining in order to detect and identify the proteins. The staining solution was prepared by dissolving 0.08 g of Coumassie blue G-250 in 200 ml of fixing solution (30 % ethanol and 10 % acetic acid in milli-Q water). The gel was allowed to stand in the staining solution for 1 hour, after which it was transferred into 200 ml of

124 destaining solution (5 % glacial acetic acid and 10 % HPLC grade methanol in distilled water), where it was allowed to stand until the background of the gel became clear. The destaining solution was changed as many times as necessary during this time.

125 2.4 Identification and Characterisation of Reference Compounds, Enzyme Substrates and Products Employed in the Study of A. alttssima Cell Suspension Cultures Studies carried out on A. altissima cell cultures include:

1. Phytochemical investigations of the alkaloid and coumarin constituents and the 2. Identification and characterisation of the methyltransferase enzymes which catalyse the méthylations of hydroxylated canthin-6-one alkaloids and coumarins.

These studies involved the use of chromatographic and spectroscopic techniques.

2.4.1 Chromatographic Techniques Chromatographic techniques employed in the identification and characterisation of the reference compounds, enzyme substrates and products include:

1. Analytical TLC, in which the Rf values of these compounds were used for their identification. 2. HPLC, in which the retention times of these compounds were used for their identification.

2.4.1.1 TLC of Canthin-6-one Alkaloids The Rf values of canthin-6-one alkaloids used were determined and presented in Table 2-12.

126 TABLE 2-12 Rf values of Canthin-6-one Alkaloid standards

Canthin-6-one Rf Value in Each Solvent System Alkaloid EtoAc CH.CLrMeOH CHCljrMeOH CHCl3:MeOH 80:20 80:20 95:5 Canthin-6-one 0.28 0.81 0.69 0.60 1 -hydroxy 0.09 0.53 0.50 0.22 canthin-6-one 1-methoxy 0.22 0.9 0.71 0.62 canthin-6-one 10-hydroxy 0.21 0.82 0.64 0.38 canthin-6-one 10-methoxy 0.18 0.85 canthin-6-one

TLC was run on slica gel 60F 254 plates, using the stated solvent systems.

2.4.1.2 TLC of Coumarins The Rf values of coumarins used were determined and presented in Table 2-13.

TABLE 2-13 Rp Values of Coumarin Standards

Coumarin Rf Value Fluorescence in UY light (365 nm) Aesculetin 0.14 bright blue Scopoletin 0.42 very bright violet Isoscopoletin 0.38 bright violet Fraxetin 0.30 dull yellow Isofraxidin 0.44 dull purple Fraxidin 0.47 dull blue Scoparone 0.68 bright purple

TLC was run on a slica gel 60F 254 plate, using ethyl acetate as the solvent system.

127 2.4.1.3 HPLC of Canthin-6-one Alkaloids The retention times of canthin-6-one alkaloids studied were determined and presented in Table 2-14.

TABLE 2-14 Retention Times of Canthin-6-one Alkaloid Standards

Canthin-6-one Alkaloid Mobile Phase Retention Time (minutes) 1 -Hydroxycanthin-6-one 55 % Methanol 10 Canthin-6-one 65 % Methanol 17 1 -Methoxycanthin-6-one 65 % Methanol 29

HPLC was run on a reversed phase column, using the stated mobile phases.

2.4.1.4 HPLC of Coumarins The retention times of the coumarins studied were determined and presented in Table 2-15.

TABLE 2-15 Retention Times of Coumarin Standards

Coumarin Retention Time (minutes) Aesculetin 17 Scopoletin 22 Isoscopoletin 22 Fraxetin 51 Isofraxidin 29 Fraxidin 26 Scoparone 8

HPLC was run on a reversed phase column, using 40 % methanol as the mobile phase.

128 2.4.2 Spectroscopic Techniques Spectroscopic techniques involved in the identification of the reference compounds, enzyme substrates and products include:

1. Fluorescent spectroscopy 2. UV spectroscopy 3. Mass spectroscopy 4. Proton nmr spectroscopy

2.4.2.1 Fluorescent Spectra of l-hydroxycanthin-6-one 1-hydroxycanthin-6-one has a bright purple fluorescence when dissolved in methanol. The fluorescent spectrum of l-hydroxycanthin-6-one was determined and the emission and excitation wavelengths recorded as 438 and 403 nm respectively. The fluorescent detector which was set at this emission and excitation wavelengths was used for the detection of 1 -hydroxycanthin-6-one during the HPLC determination described in Sub-Section 22.2.2.

2.4.2.2 UV, MS and NMR Spectra of Canthin-6-one Alkaloid Standards used as Reference Compounds, Enzyme Substrates and Products

Canthin-6-one UV (MeOH) 250, 259, 267, 300, 346, 360, 378 nm. MS, M/Z(%) 220(M\ 100), 192(52), 166(5), 165(7), 164(7), 139(7), 114(12), 96(10). 'H-nmr (250 MHz), 5 7.00(1H, d, J=10Hz, H-5), 7.55(1H, m, J=8Hz, H-10), 7.73(1H, m, J=8Hz, H-9), 8.00(1H, d, J=5Hz, H-1), 8.05(1 H, d, J=10Hz, H-4), 8.14(1H, d, J=8Hz, H-11), 8.70(1H, d, J=8Hz, H-8), 8.85 ppm(lH, d, J=5Hz, H-2).

1 -Hydroxycanthin-6-one UV (MeOH) 265, 288, 335, 345, 362, 382 nm.

129 MS, M/Z(%) 236(M\ 100), 208(65), 181(17), 153(42), 128(26), 103(22). 'H-nmr (250 MHz), 5 6.68(1 H, d, J=10Hz, H-4), 7.43(1 H, m, J=8Hz, H-10), 7.53(1 H, m, J=8Hz, H-9), 7.82(1 H, d, J=10Hz, H-5), 8.15(1H, d, J=10Hz, H-11), 8.20(1 H,s, H-2), 8.53 ppm(lH, d, J=10Hz, H-8).

1 -Methoxycanthin-6-one UV (MeOH) 259, 270, 280, 346, 370, 378 nm. MS, M/Z(%) 250(MM00%), 235(9), 222(9), 220(8), 208(6), 207(37), 192(5), 180(12), 179(11), 152(11). 'H-nmr (250 MHz), Ô 4.28(3H, s, I-OCH3), 6.87(1H, d, J=10Hz, H-5), 7.45(1H, m, J=8Hz, H-10), 7.68(1 H, m, J=8Hz, H-9), 7.99(1H, d, J=10Hz, H-4), 8.25(1H, d, J=10Hz, H-11), 8.52(1 H, S, H-2), 8.70 ppm(lH, d, J=10Hz, H-8).

10-Hydroxycanthin-6-one UV (MeOH) 269, 275, 288, 304, 312, 352 nm. MS, M/Z 236(MM00%), 209(14), 208(99), 179(26), 153(15), 127(13), 126(13), 118(7), 104(20), 77(12), 76(24), 75(20), 74(12), 62(25) and 52(18). 'H-nmr (60 MHz), 06.94(1 H, d, J=9.9Hz, H-5), 6.99(1 H, dd, J=2.3,8.5Hz, H-9), 7.99(1 H, d, J=2.3Hz, H-11), 8.10(1H, d, J=9.9Hz, H-4), 8.11(1H, d, J=4.9Hz, H-1), 8.14(1H, d, J=8.5Hz, H-8) and 8.74 ppm(lH, d, J=4.9Hz, H-2).

10-Methoxycanthin-6-one UV (MeOH) 212, 232, 266, 274, 310, 352 nm. MS, M/Z 250(M\ 100%), 249(14), 222(10), 221(20), 220(15), 207(8), 192(14), 179(19), 153(11), 125(11), 111(12) and 97(19). 'H-nmr (60 MHz) Ô 3.91(3H, S, IO-OCH3), 6.83(1H, d, J=9.8Hz, H-5), 6.91(1H, dd, J=2.4, 8.5Hz, H-9), 7.65(1H, d, J=5.0Hz, H-1), 7.74(1 H, d, J=8.5Hz, H-8), 7.89(1 H, d, J=9.8Hz, H-4), 7.97(1 H, d, J=2.4Hz, H-11) and 8.65ppm(lH, d, J=5.0Hz, H-2).

130 2 4.2.3 UV, MS and NMR Spectra of Coumarin Standards used as Reference Compounds, Enzyme Substrates and Products

Aesculetin (6,7-dihydroxycoumarin) UV (MeOH) 260, 290, 300, 350 nm. MS, M/Z 178(MM00%), 171(3), 150(99), 137(6), 132(5), 129(7), 121(7) 111(12), 104(6), 98(22), 94(12), 83(28), 79(11), 74(22), 70(46), 66(12), 58(35) and 55(42). 'H-nmr (400 MHz) 05.92(1H, d, J=9Hz, H-3), 7.54(1H, d, J=9Hz, H-4), 6.74(1H, S, H-5), 6.52 ppm(lH, S, H-8).

Scopoletin (7-hydroxy,6-methoxycoumarin) UV (MeOH) 229, 252, 260, 297, 344 nm. MS M/Z 192(MM00%), 177(61), 165(20), 149(62), 121(24), 93(5), 79(14), 75(4), 69(66), 65(12). 'H-nmr(400 MHz) 63.93(3H, S, 6-OCH3), 6.25(1H, d, J=9.4Hz, H-4), 6.87(1H, S, H-5), 6.88(1 H, S, H-8) and 7.66 ppm(lH, d, J=9.4Hz, H-3).

Isoscopoletin (6-hydroxy,7-methoxycoumarin) UV 260, 300, 350 nm. MS M/Z 192(MM00%), 177(11), 165(19), 149(64), 121(12), 79(17), 77(4), 69(24), 65(6). 'H-nmr(400 MHz) 03.97(3H, S, 6-OCH3), 6.27(1H, d,J=9.4Hz, H-4), 6.85(1H, S, H- 5), 6.95(1H, S, H-8) and 7.64 ppm(lH, d, J=9.4Hz, H-3).

Scoparone (6,7-dimethoxycoumarin) UV (MeOH) 240, 260, 350 nm. MS, M/Z 206(MM00%), 191(41), 178(17), 163(29), 135(18), 107(17), 69(28). 'H-nmr(250 MHz) 67.62(1H, d, J=10Hz, H-4), 6.82(2H, S, H-5 and H-8), 6.24(1 H, d, J=10Hz, H-3), 3.96(3H, S, 6-OCH3), 3.95(3H, S, 7-OCH3).

131 Fraxetin (7,8-dihydroxy ,6-methoxycoumarin) UV (MeOH) 260, 350 nm MS M/Z 208(MM00%), 193(32), 181(15), 166(12), 152(5), 137(18), 109(22), 96(5), 81(15), 69(4), 64(7), 53(19). 'H-nmr (400 MHz) Ô3.96(3H, S, 6-OCH3), 6.218(1H, d, J=9.4Hz, |H-4), 6.56(1 H, S, H-5), 7.687 ppm(lH, d, J=9.4Hz, H-3).

Fraxidin (8-hydroxy,6,7-dimethoxycoumarin) UV X^,,(MeOH) 320 nm. MS M/Z 222(MM00%), 207(27), 194(3), 189(4), 179(7) 162(6), 151(9), 133(11), 123(32), 108(9), 95(8), 79(9), 69(4) , 63(5). 'H-nmr (400 MHz) Ô3.911(3H, S, 6-OCH3), 3.96(3H, S, 7-OCH3), 6.35(1H, d, J=9.4Hz, H-4), 6.58(1 H, S, H-5), 7.75 ppm(lH, d, J=9.4Hz, H-3).

Isofraxidin (7-hydroxy,6,8-dimethoxycoumarin) UV X^,, (MeOH) 350 nm. MS M/Z 222(MM00%), 207(31), 194(21), 179(24), 151(16), 123(24), 95(22). 'H-nmr (400MHz) Ô3.84(3H, S, 8-OCH3), 3.90(3H, S, 6-OCH3), 6.24(1H, d, J=10Hz, H-3), 7.01(1H, S, H-5), 7.92 ppm(lH, d, J=10Hz, H-4).

132 3. RESULTS

133 3.1 Development of an Enzyme Assay for A. altissima Methyltransferase Enzymes and the Characterisation of Products of the Méthylation Reaction Preliminary experiments were designed in order to obtain an efficient and accurate enzyme assay procedure for the hydroxylated canthin-6-one and coumarin methyltransferase enzymes of A. altissima.

3.1.1 Development of Enzyme Assay The assay procedure used for 1 -hydroxycanthin-6-one methyltransferase as well as coumarin methyltransferase (described in detail in Sub-Section 2.3.3), involved obtaining an incubation mix of 130 pi in volume, comprising: the enzyme solution from A. altissima cell suspension cultures; the required hydroxylated substrate and "*C- SAM. This was followed by incubation, termination of the enzyme reaction and extraction of the methylated products for radioactivity determination in a scintillation counter. Preliminary experiments were carried out, to determine the optimum conditions for enzyme assays, i.e. optimum protein levels, temperature, pH and incubation time as well as saturating levels of the substrates. The results obtained and the methodology involved in these experiments are discussed in detail in Sub-Section 3.4.1.

3.1.2 Recovery of Methylated Products These experiments were performed for two main purposes; firstly to ensure that during the enzyme assay procedure (described in Sub-Section 2.3.3), most of the product formed during the méthylation reaction, was successfully extracted into the organic phase (800 pi of toluene used for product extraction). Secondly experiments were carried out to prove that it is possible to accurately transfer the extracted product into a scintillation vial prior to counting in a scintillation counter. These experiments were carried out on the partially purified enzyme solution i.e. the desalted (NH4)2S0 ^ precipitate from A. altissima cell suspension cultures.

134 3.1.2.1 Determination of Levels of l-methoxycanthin-6-one Recovered During Product Extraction These experiments involved the preparation and subsequent assay for enzyme activity of 10 routine incubation mixes in Eppendorf tubes, as described in Sub- Section 2.3.3.1. Each incubation mix was incubated, after which the reaction was terminated and product extraction was subsequently carried out as described in Sub- Section 2.3.3.3. Product extraction involved the addition of 800 pi of toluene to each Eppendorf tube and |800 pi of this was transferred into a scintillation vial for radioactivity counting. Enzyme activity measured in dpm, was recorded for each of the ten assays as shown in Table 3.1-10.

TABLE 3.1-10 Determination of the Levels of l-Methoxycanthin-6-one Recovered by Product Extraction during Enzyme Assay

Incubation Enzyme Activity in Extracted Products (dpm) % of Total Mix Enzyme Assayed for Activity Enzyme r* Extraction 2"'* Extraction 3'*’ Extraction Recovered Activity after P ‘ Extraction

1 4585.91 157.74 91.46 94.8

2 4176.14 105.78 106.07 95.2

3 4671.37 143.57 84.96 95.3

4 4733.50 176.33 97.11 94.5

5 4187.04 117.62 93.26 95.2

6 4312.95 124.06 97.83 95.1

7 4270.27 121.19 92.49 95.2

8 4376.29 118.58 90.77 95.4

9 4505.94 155.39 101.22 94.6

10 4470.02 136.12 98.56 95.0

Assays for 1-HMT activity were done under routine conditions using the reconstituted (NH4)2S04 precipitate from A. altissima cell suspension cultures.

135 Two subsequent extractions were also carried out on each of these 10 incubation mixtures, by adding another |800 pi volume of toluene to each of the incubation mixtures and subsequently extracting and removing |8GQ pi for scintillation counting. The enzyme activity recovered during each of the 2 subsequent extractions were recorded for all 10 incubation mixes as shown in Table 3.1-10. The percentage recovery of enzyme activity from the first extraction was computed for each of the 10 enzyme assays done and the results showed that for all the 10 enzyme assays, an average of 96 % of the methylated product was recovered in the first extraction, (mean ± S.D = 96.03 ± 0.81). These results confirmed the efficiency of the extraction procedure used.

3.1.2.2 Determination of the Reproducibility of the Product Extraction Procedure These experiments were carried out to demonstrate that it is possible to accurately transfer the extracted product (present in 800 pi of toluene) into a scintillation vial for scintillation counting. Experiments carried out were aimed at showing that most of the toluene extract could be precisely transferred from the Eppendorf tube (in which the assay and product extraction were carried out, as described in Sub-Section 2 3 3 3 ) into the scintillation vial without causing appreciable disturbance to the aqueous phase. Disturbance of the aqueous phase is undesirable as it would result in the contamination of organic phase with background radioactivity. These experiments were carried out visually, i.e. by observing the Eppendorf vials containing the incubation mixture (aqueous phase) and toluene (organic phase) for a disturbance of the aqueous phase during the removal of the organic phase. The results demonstrated that it is possible to accurately transfer only 6(X) pi of the 800 pi toluene used for product extraction. In order to make up for this discrepancy, the shortfall ratio of 600 pi : 800 pi was taken into consideration whilst calculating enzyme activity, where the calculated enzyme activity was upgraded to reflect the residual radioactivity in the 200 pi of toluene left behind during the extraction procedure. The equation used for calculating

136 enzyme activity is given in Sub-Section 2.33.4 (Equation 2-100).

3.1.3 Determination of Background Radioactivity These experiments were carried out to show that during the enzyme assay procedure (Sub-Section 2.3.3.3), significant levels of background radioactivity (residual radioactivity unassociated with the methylated product), were not carried through from the aqueous phase into the organic phase, during product extraction. The experimental procedure involved comparing enzyme activity obtained from a normal or routine assay, with that of blank or 0 % control assays in which the hydroxylated substrate had been excluded. This was achieved by preparing 3 incubation mixtures for routine enzyme assays; each containing the partially purified enzyme solution as well as the enzyme substrates ‘"^C-SAM and l-hydroxycanthin-6- one (as previously described in Sub-Section 2.3.3.1). 10 blank or 0 % control assays were also prepared, which contained the partially purified enzyme solution and SAM but the hydroxylated substrate was excluded. These incubation mixes (i.e. for the normal and control enzyme assays) were then incubated and the enzyme activity determined as described in Sub-Section 2.3.3.3. The average enzyme activity in dpm obtained from the 3 normal assays were compared with the enzyme activity in dpm obtained from each of the 10 blank assays and the results presented in a tabulated form (Table 3.1-11). Levels of radioactivity obtained during formation of the labelled product in the normal or routine assays, served as a yard stick for checking that if background radioactivity was carried through during the enzyme assay for 1-HMT activity, the levels were acceptable. Since the blank assays contained no hydroxylated substrate, it was expected that formation of the labelled product of the enzyme assay would be non-existent. In an ideal situation, no radioactivity would be present in the extracted product from these assays. Radioactivity was however carried through into the extracted product of these blank assays. This was regarded as background radioactivity, and in subsequent experiments was subtracted from the raw detected dpm.

137 TABLE 3.1-11 Determination of the Level of Background Radioactivity

Incubation Mix Enzyme Enzyme Activity in Enzyme Activity in Assayed for Activity in the 0 % Control the Control Expressed Enzyme Activity the Normal Incubation as a % of the normal Incubation mix (dpm) incubation mix mix (dpm)*

1 7792 705 9.04

2 716 9.18

3 780 10.01

4 743 9.53

5 739 9.48

6 747 9.58

7 754 9.68

8 791 10.15

9 774 9.93

10 724 9.33

Legend: * Average of triplicates

Assays for 1-HMT activity were done under routine conditions using the reconstituted (NH4)2S04 precipitate from A. altissima cell suspension cultures.

From these results, (Table 3.1-11) it was observed that in each case the normal assay was about 10 times greater than the blank assay, (mean ± S.D = 9.591 ± 1.091). This distinct difference in the levels of actual and background radioactivity was considered as being acceptable.

3.1.4 Determination of the Reproducibility of the Assay Procedure Experiments were carried out to ensure that the assay procedure employed for the determination of enzyme activity was reproducible. The experimental procedure involved the preparation of a set of 10 incubation mixes for routine assays, each in a total volume of 130 pi, as previously described in

138 detail in Sub-Section 2.3.3.1. Enzyme assays were subsequently carried out as described in Sub-Section 2.3.3.3, and enzyme activity in dpm subsequently computed for each of the incubation mixes utilised for experimentation. The results are shown in Table 3.1-12. The difference in enzyme activity obtained for each of the 10 assays when compared statistically were acceptable (mean ± S.D = 5423.8 ± 437.06). On the basis of these results it was concluded that enzyme activity obtained for each of the 10 assays were close enough to be considered as being reproducible.

TABLE 3.1-12 An investigation of the Reproducibility of the Assay Procedure Used for the 1-HMT Enzyme

Incubation Mix Assayed Enzyme Activity for Enzyme Activity (dpm) 1 5261 2 5316 3 5310 4 5369 5 5586 6 5342 7 5904 8 5437 9 5422 10 5291

Assays for 1 -HMT activity were done under routine conditions using the reconstituted (NHJ2SO4 precipitate from A. altissima cell suspension cultures.

139 3.1.5 The Sulphydryl Group Requirement Most methyltransferase enzymes require sulphydryl (SH) protecting agents for stability. It was therefore logical to presume the same would apply to the methyltransferase enzyme of A. altissima. Experiments were performed to verify this assumption. These experiments involved the determination of the effect of two sulphydryl group protecting agents i.e. p-Mercaptoethanol (p-MCE) and dithioerythritol (DTT) on stability of the methyltransferase enzyme of A. altissima. A crude dialysate of A. altissima suspension cultures was used for carrying out this experiment. The buffer for enzyme extraction i.e. homogenisation buffer A was prepared as described in Sub-Section 2.3.1, but p-MCE was omitted. This buffer was then divided into 3 batches; the first batch labelled buffer 1, contained 60 mM levels of P- MCE, the second batch labelled buffer 2, contained 10 mM levels of DTT and the third batch labelled buffer 3, contained no additives. Three, 5 g batches of harvested suspension cultures of A. altissima were weighed out and labelled as cell culture batches 1, 2 and 3. A crude dialysate of the first cell culture batch was obtained as described in Sub-Section 2.3.2, using buffer 1, for enzyme extraction. Crude dialysates of the second and third cell culture batches were similarly prepared using buffers 2 and 3 respectively. The crude dialysates obtained from each batch of cultures were individually filtered through a nylon bolting cloth, to obtain three enzyme preparations. Assays for enzyme activity were carried out on the three enzyme preparations, in triplicate and the results presented in Figure 3.1-1, which shows that there was almost complete loss of methyltransferase activity in the absence of sulphydryl group protecting agents. These results also showed that there was no significant difference between the use of p-MCE and DTT as enzyme activity remained at similar levels in the presence of both compounds as shown in Figure 3.1-1. p-MCE was subsequently selected for use in enzyme assays, as it is cheaper.

140 FIGURE 3.1-1 The Investigation of the Requirement of A. altissima Methyltransferases for Sulphydryl Group Protection

4000 -

S- 3200 -

> 2400 -

1600 - c UJ 800 -

8-MCE DTT CTRL

Additives Containing SH Groups The assay was done under routine conditions using active fractions from the crude dialysate from A. altissima cell suspension cultures. SH protecting agents investigated include: |3-Mercaptoethanol (J3-MCE) and Dithioerythritol (DTT). The control assay (CTRL) had no SH group containing additive.

141 3.1.6 Product Identification

3.1.6.1 Identification of l-Methoxycanthin-6-one Experiments were carried out to verify the identity of the product of 1- hydroxycanthin-6-one méthylation by the 1-HMT enzyme (l-methoxycanthin-6-one). The méthylation reaction investigated is represented in Equation 3.1-100.

1-hydroxy-C-6-one + '"^C-SAM —> ^"^C-1 -Methoxy-C-6-one + SAH Equation 3.1-100.

The experimental procedure involved in the identification of 1 -methoxycanthin- 6-one is described in Sub-Section 2.3.3.5. This method involved running a TLC chromatogram (shown in Figure 3.1-2) of the extracted product and comparing its migratory characteristics with that of standard l-methoxycanthin-6-one. Other small molecules had been removed during the enzyme preparation and only l-hydroxycanthin-6-one had been added to the reaction mix; therefore 1- methoxycanthin-6-one was the expected méthylation product. In order to aid visualisation of the methylated product, cold l-methoxycanthin-6-one was added to the reaction mix after incubation and prior to the extraction of the methylated product. A positive identification was made when the Rf value of the product corresponded with the Rf value of the standard 1 -methoxycanthin-6-one. The identity of the methylated product was confirmed by examining the product band for radioactivity. This was achieved by cutting up the column of the chromatogram containing the extracted product into 1 cm bands (as described in Sub-Section 2.3.3.5), and comparing radioactivity levels in each band with that of the previously determined 100 % control, which had an average enzyme activity of 7410 dpm. The chromatogram which was traced before it was cut up into bands is shown in Figure 3.1-2). This chromatogram showed that of all the bands examined for radioactivity only the band corresponding to l-methoxycanthin-6-one had significant levels of radioactivity concentrated in it. This results indicate that most of the ‘"^C-

142 label is transferred from ^"^C-SAM to l-hydroxycanthin-6-one, thereby forming ^"^C-1-

FIGURE 3.1-2 Identification of the Product of l-hydroxy-C-6-one Méthylation (by the 1-HMT Enzyme), Using TLC Techniques.

Band Enzyme TLC Chromatogram Number Activity (dpm)* Extracted Standard Standard Product 1-methoxy- 1-hydroxy- C-6-one C-6-one 16 66 15 52 14 504 C ) C ) 13 71 12 77 11 75 10 59 9 65 c : ) 8 71 7 64 6 47 5 50 4 52 3 53 2 63 1 86 0

Legend: * present in each band of the chromatogram from the extracted product. (Cold l-methoxycanthin-6-one had been added to the product prior to extraction). Chromatography was carried out on a silica gel 60F-254 plate, in a solvent system of CH2CI2 : methanol, 80 : 20.

143 methoxycanthin-6-one. Levels of radioactivity corresponding to the methylated product (504 dpm), however failed to correlate with radioactivity levels in the control sample (7410 dpm). Controls showed that this discrepancy could be attributed to quenching caused by the presence of the silica gel disc in the scintillation vial, during the determination of levels of radioactivity in the extracted product.

3.1.6.2 Identification of the Methylated Coumarins The identities of the methylated products of the coumarin methyltransferase enzymes were verified for the two sequences of coumarin méthylation investigated. These sequences are schematically represented below with full structural details given in Sub-Section 3.5.2, Figure 3.5-1.

Sequence 1 [-^Scopoletin Aesculetin — ►6,7-dimethoxycoumarin ►Isoscopoletin ... Equation 3.1-101

Sequence 2

r-»Fraxidin Fraxetin —>6,7,8-trimethoxycoumarin —^Isofraxidin ... Equation 3.1-102

144 3.1.6.2.1 Identification of the Products of Aesculetin Méthylation The méthylation reaction investigated is represented in Equation 3.1-103

Aesculetin + "^C-SAM —> '"‘C-Scopoletin ± ‘"‘C-Isoscopoletin + SAH Equation 3.1-103 The experimental procedure involved is described in detail in Sub-Section 2.3.3.5. A TLC chromatogram of the extracted product was run in ethyl acetate as described in Sub-Section 2.3.3.5, and the relative movement of bands compared with those of standard scopoletin and isoscopoletin. Other small molecules had previously been removed from the enzyme preparation, and only aesculetin was added to the incubation mix, so the expected products were scopoletin and or isoscopoletin. Cold scopoletin and isoscopoletin (the possible products of aesculetin méthylation) were therefore added to the incubation mix after incubation and prior to the extraction of the methylated product, to aid visualisation of the methylated product. The results presented in Figure 3.1-3, showed that on running the TLC, the product band produced three bands; two of which corresponded with both scopoletin and isoscopoletin as shown in Figure 3.1-3, and the other was not identified. The solvent system used in developing this chromatogram (Ethyl acetate), separated the isomers scopoletin and isoscopoletin, hence it was possible to determine which of these two isomers was actually formed during the méthylation of aesculetin by the A. altissima CMT enzyme. Further investigation of the identity of the product involved cutting up the column of the chromatogram containing the extracted product into 1 cm bands and comparing the radioactivity levels in each band with that of the previously determined 100 % control, which had an average enzyme activity of 6024 dpm. The chromatogram which was traced before it was cut up into bands is shown in Figure 3.1-3. This chromatogram showed that of all the bands examined, only the band corresponding to scopoletin had significant levels of radioactivity concentrated in it i.e. 2813 dpm. These results further indicate that most of the '"^C-label is

145 FIGURE 3.1-3 Identification of the Products of Aesculetin Méthylation (by CMT Enzyme), Using TLC Techniques.

Band Enzyme TLC Chromatogram Number Activity (dpm)* Extracted Standard Standard Standard Product Scopoletin Isoscopoletin Aesculetin

12 93 11 46 10 70 o 9 105 8 110 7 135 6 2813 o n 5 130 4 136 3 122 O 2 66 1 151 0

Legend: * present in the chromatogram of the extracted product. (Cold scopoletin and isoscopoletin had been added to the product prior to extraction). Chromatography was carried out on a silica gel 60F-254 plate, in a solvent system of ethyl acetate. transferred from ^"^C-labelled SAM to aesculetin, thereby forming ^"^C-Scopoletin. Levels of radioactivity, corresponding to the methylated product (2813 dpm) however failed to correlate with radioactivity levels in the control (6024 dpm). Controls showed that this discrepancy can be attributed to quenching caused by the presence of the silica gel disc in the scintillation vial, during the determination of levels of radioactivity in the extracted product.

146 These results indicate that the product of aesculetin méthylation by the CMT enzyme of A. altissima is scopoletin. Experiments carried out for purposes of identification of the methylated product were not extended to the dimethylation products, however preliminary experiments were carried out to investigate the formation of scoparone (6,7- dimethoxycoumarin), as shown in sequence 1 for coumarin méthylation (Sub-Sections 3.1.6.2 and 3.5.2). These experiments which are described in detail in Sub-Section 3.5.2, involved assaying the partially purified CMT enzyme, using aesculetin, scopoletin and isoscopoletin as substrates. The results obtained (presented in Table 3.5-10, Sub-Section 3.5.2), showed that significant méthylation of isoscopoletin was observed, though low in comparison to aesculetin méthylation, whilst scopoletin méthylation was non-existent. These results indicate that CMT is an enzyme which methylates aesculetin at position C-6, but not C-7. These results, together with the fact that ‘"‘C-isoscopoletin was not detected in these cultures suggest that scoparone is not present in cell cultures of A. altissima.

3.1.6.2.2 Identification of the Products of Fraxetin Méthylation The méthylation reaction investigated is represented in Equation 3.1-104

Fraxetin + '"^C-SAM —> '"^C-Isofraxidin ± '"^C-Fraxidin + SAH Equation 3.1-104 The experimental procedure employed is described in detail in Sub-Section 2.3.3.5. A TLC chromatogram of the extracted product was run in ethyl acetate and the relative movement of bands compared with those of standard fraxidin and isofraxidin . Other small molecules had been removed during the enzyme preparation and only fraxetin had been added to the incubation mix, so the expected products were isofraxidin and or fraxidin . Therefore in order to aid visualisation of the methylated product, cold isofraxidin and fraxidin were added to the incubation mix after

147 FIGURE 3.1-4 Identification of the Products of Fraxetin Méthylation (by CMT Enzyme), Using TLC Techniques.

Band Enzyme TLC Chromatogram Number Activity (dpm)* Extracted Standard Standard Standard Product Fraxidin Isofraxidin Fraxetin 12 81 11 102 10 96 O 9 58 8 142 7 348 D 6 3943 O 5 248 4 167 CD 3 105 2 101 1 131 0

Legend: * present in the chromatogram of the extracted product. (Cold fraxidin and isofraxidin had been added to the product prior to extraction). Chromatography was carried out on a silica gel 60F-254 plate, in a solvent system of ethyl acetate. incubation and prior to the extraction of the methylated product. The results presented in Figure 3.1-4, showed that on running the TLC, the product band was separated into 3 bands, 2 of which corresponded with isofraxidin^ and fraxidin, respectively as shown in Figure 3.1-4. The third band was not identified. Further investigation of the identity of the product, involved cutting up the

148 portion of the chromatogram containing the product into 1 cm bands and comparing radioactivity levels in each band with that of the previously determined 100 % control which had an average enzyme activity of 12,613 dpm (see Figure 3.1-4). The chromatogram which was traced before it was cut up into bands is shown in Figure 3.1-4. This chromatogram showed that of all the bands examined, the most significant levels of radioactivity (3943 dpm) was concentrated in one of the product bands (the band with an Rf value similar to isofraxidin ). A lower level of radioactivity (348 dpm) was concentrated in the other product band (that which had an Rf value similar to Fraxidin ), as shown in Figure 3.1-4. These results show that isofraxidin contained most of the label transferred from "‘C-SAM to fraxetin, thereby forming ‘"‘C-isofraxidin as the major product. Fraxidin contained a lower level of the label. This may indicate that ‘"^C- fraxidin was formed as the minor product, however it is more likely to be as a result of the carry through to fraxidin , of the radioactivity associated with isofraxidin . This effect can be seen in the two product bands on either side of the band corresponding to isofraxidin . Experiments carried out for purposes of identification of the methylated product were not extended to the dimethylation products, however preliminary experiments were carried out to investigate the dimethylation of fraxetin to form 6,7,8- trimethoxycoumarin, (as shown in sequence 2 for coumarin méthylation, Sub-Sections 3.1.6.2 and 3.5.2). These experiments which were carried out as described in Sub- Section 3.5.2, involved assaying the partially purified CMT for activity, using fraxetin, isofraxidin and fraxidin as substrates. Results presented in Table 3.5-11, of Sub- Section 3.5.2, showed that significant méthylation of fraxidin occurred though low in comparison to fraxetin méthylation, whilst méthylation of isofraxidin was non existent. These results suggest that CMT is an enzyme which will methylate fraxetin specifically at C-8, but not at C-7. These results, together with the fact that very low levels of the ‘"^C label corresponding to fraxidin were detected in these cultures, suggest that 6,7,8- trimethoxycoumarin is not present in cell cultures of A. altissima.

149 3.2 Time Course Studies of A. altissima Cell Suspension Cultures Several cell lines of A. altissima cell suspension cultures have been established and maintained in our laboratories (Anderson et al., 1983; Hay, 1987; Anderson et al., 1987 a,b). These cultures were found to produce relatively high yields of the canthin- 6-one alkaloids: canthin-6-one, l-hydroxycanthin-6-one and l-methoxycanthin-6-one. They also produced significant levels of the coumarins scopoletin and isofraxidin (Hay, 1987). Methyltransferase enzymes are considered as possible alternatives to chemical methods of carrying out méthylation reactions. Cultures of A. altissima served as a model system for studying the activity of an 0-methyltransferase enzyme, which could be potentially useful for carrying out general méthylation reactions. In this study the methylating ability of A. altissima cell cultures was studied in detail with respect to both méthylation of alkaloids and coumarins. To investigate the methylating potential of A. altissima cell suspension cultures, detailed time course studies were carried out on two of the A. altissima cell lines developed and maintained in our laboratories. Cell line 1 which had 1- methoxycanthin-6-one as the major constituent and cell line 2 which had canthin-6-one as the major constituent. Studies of these cell lines of A. altissima involved the investigation of their growth, alkaloid and coumarin production patterns as well as the alkaloid and coumarin methyltransferase enzyme activities. Cell suspension cultures of A. altissima were maintained under conditions previously described in Sub-Section 2.1.3. The suspension cultures used for these experiments were initiated by aseptically harvesting and transferring 3 g fresh weight of inoculum into flasks containing 40 ml of Murashige and Skoog liquid medium. These cultures were maintained for a period of at least 30 days and cultures were harvested in duplicate on alternate days throughout the growth cycle. The harvested cultures were assayed qualitatively and quantitatively for the presence of canthin-6-one alkaloids and coumarins and also for methyltransferase enzyme activity. This analysis involved the extraction of canthin-6-one alkaloids and coumarins by methods previously described in Sub-Section 2.2.1, and their subsequent identification by TLC techniques described in Sub-Sections 2.2.2, 2.4.1 and 3.1.6.

150 These constituents were then quantified, by using the reversed phase HPLC techniques described in Sub-Section 2.2.22. The harvested samples were also assayed for alkaloid and coumarin methyltransferase activity. This was done by extracting the methyltransferase enzyme from harvested cell cultures, using enzyme extraction techniques previously described in Sub-Section 2.3.2, followed by the removal of aromatic compounds with Amberlite XAD-2 resin as described in Sub-Section 2.3.4.1. The extracted enzyme was assayed for alkaloid and coumarin methyltransferase activity, using assay techniques described in Sub-Section 2.3.3.3.

3.2.1 Growth, Canthin-6-one Alkaloid Production and Corresponding Canthin-6- one Alkaloid Methyltransferase Activity in Cell Suspension Cultures of A.altissima

3.2.1.1 Cell Line 1 3.2.1.1.1 Growth of Cells The growth pattern of A. altissima cell suspension cultures (cell line 1) is shown in Figure 3.2-1(c). The cells multiplied quite rapidly after day 12, undergoing a five fold increase in fresh weight over the growth cycle. The growth cycle occurred as an archetypal sigmoid pattern with clearly defined lag, exponential linear and stationary phases occurring at around days 0 to 12, 12 to 14, days 14 to 22 and days 24 to 28 respectively.

3.2.1.1.2 Alkaloid Production Studies of the alkaloid production pattern of cell line 1 demonstrated as shown in Figure 3.2-1 (a), that 1-methoxycanthin-6-one was the major alkaloid constituent, produced at levels of 5 to 70 mg . g . dry wt. of cells'*. Lower levels of canthin-6-one (10 to 30 mg . g . dry wt. of cells'*) and l-hydroxycanthin-6-one (2 to 3 mg . g. dry wt. of cells'*) were produced. Results presented in Figure 3.2-1 (a) showed that the cells commenced production of significant levels of l-methoxycanthin-6-one at about day 16.

151 FIGURE 3.2-1 Time Course Study of Alkaloid Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell line 1)

(/I 75 - a. Alkaloid levels o 1 —Methoxy—C—6-one ^ Canthin-6-one 60 - ■ 1 —Hydroxy—C—6—one

■a 4 5 - d)

30 - O)

-Q ir

b. Alkaloid methyltransferase activity using 20000 - O 1 -Hydroxy-C-6-one as substrate g- 16000 -

12000 - ta 8000 -

LJJ 4000 -

•-5 15 - 0 . Cell growth

12 -

0 6 12 18 24 30 Time (Days)

152 1-methoxycanthin-6-one production subsequently rose steadily reaching maximum levels of 70 mg . g dry wt. of cells ' at about day 21. Canthin-6-one alkaloid production showed a less significant increase to levels oscillating between 15 to 30 mg . g dry wt. of cells'* at between days 16 to 21 of the growth cycle. 1- hydroxycanthin-6-one alkaloid production was the least of the 3 alkaloids investigated and remained fairly constant at levels of 2 to 3 mg . g dry wt. of cells'*, throughout the growth cycle.

3.2.1.1.3 1-HMT Activity The 1-HMT enzyme was operative throughout the growth cycle as shown in Figure 3.2-1 (b), with significant levels of enzyme activity occurring between days 6 to 24. Two peaks of activity occurred from days 6 to 12 and days 20 to 22 of the growth cycle. A slight dip in activity occured in between the two peaks.

3.2.1.2 Cell Line 2 3.2.1.2.1 Growth of Cells Cell line 2 of A. altissima cell suspension cultures, underwent fairly rapid multiplication yielding a five fold increase in fresh weight over the growth cycle. This growth cycle, had a shorter lag phase than cell line 1. The lag phase occured from days 0 to 8, after which the exponential, linear and stationary phases occurred. In this cell line i.e. Figure 3.2-2(c), the exponential, linear and stationary phases were less clearly defined than cell line 1. It is however clear that in this cell line the cells entered into their stationary phase a little earlier than cell line 1. The stationary phase occured between days 20 and 28.

3.2.1.2.2 Alkaloid Production Studies of the alkaloid production pattern of cell line 2 demonstrated as shown in Figure 3.2-2(a), that canthin-6-one was the major alkaloid constituent produced at levels of 5 to 22 mg . g dry wt. of cells'*. These cells also produced 1- methoxycanthin-6-one and l-hydroxycanthin-6-one as minor constituents at levels of

153 FIGURE 3.2-2 Time Course Study of Alkaloid Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell line 2]

25 - a. Alkaloid levels o 1 —Methoxy—C—6—one ^ Canthin-6-one ■ 1—Hydroxy—C—6—one 20 -

15 - O)

10 - o>

■o 5 0 e

20000 - . Alkaloid methyltransferase activity using o 1-Hydroxy-C-6-one as substrate

^ 1 6 0 0 0 -

12000 -

8 0 0 0 -

4 0 0 0 - LU

E 0 ------10.0 - c. Cell growth

8.0 -

6.0 -

cn 4.0 -

2.0 - u_ 0.0 O 0 16 24 328 40 Time (Days)

154 1 to 6 mg . g dry wt. of cells ' and 1 to 2.5 mg . g dry wt. of cells ' of cells respectively. Figure 3.2-2(a) shows the alkaloid production pattern of cell line 2. 1 -methoxycanthin-6-one levels remained very low until about day 16, when a slight increase to levels ranging from 3 to 6 mg . g dry wt. of cells'', occurred until day 34 of the growth cycle. The cells commenced production of significant levels of canthin- 6-one at about day 16. Canthin-6-one levels rose steadily after that, reaching maximal levels of 22 mg . g dry wt. of cells ' at about day 28 of the growth cycle. 1- hydroxycanthin-6-one production in cell line 2, as with cell line 1, was the lowest of the three canthin-6-one alkaloids, l-hydroxycanthin-6-one levels remained quite constant at 1 to 2.5 mg . g dry wt. of cells'' throughout the growth cycle, without any obvious peaks of activity.

3.2.1.2.3 1-HMT Activity The 1 -HMT activity in cell line 2 was operative throughout the growth cycle, except from days 0 to 6 and days 26 to 28 as shown in Figure 3.2-2(b). Peaks of 1- HMT activity occurred between days 12 to 20 and days 30 to 34.

3.2.2. Production of Methylated Coumarins and Corresponding Methyltransferase Activity in Cell Suspension Cultures of A. altissima Hay, 1987 and Roberts, 1991 reported the occurrence of scopoletin and isofraxidin in cell suspension cultures of A. altissima. This suggested the existence of a biosynthetic route from aesculetin to isofraxidin in cell suspension cultures of A. altissima. A similar biosynthetic route has been proposed by Brown, 1988; Dewick, 1985, 1990). The extent to which this pathway was operable in terms of production of methylated coumarins and the corresponding CMT activities was investigated in the course of these studies. Time course studies involving the investigation of coumarin levels and corresponding coumarin methyltransferase activity were carried out with cell lines 1 and 2 of A. altissima cell suspension cultures.

155 The coumarins present in identifiable levels in these cultures include scopoletin (the méthylation product of aesculetin) and isofraxidin (the méthylation product of fraxetin) in sequences 1 and 2 for coumarin méthylation respectively (Sub-Sections 3.1.6.2 and 3.5.2). The HPLC technique employed did not easily separate scopoletin from its isomer isoscopoletin (Sub-Section 22.22 and Table 2-15, Sub-Section 2.4.1). It was therefore not possible to make conclusive statements on the actual identity of the product of aesculetin méthylation by using HPLC techniques. However the TLC system employed (Sub-Section 2.2.2.1 and Table 2-13, Sub-Section 2.4.1) separated these two isomers. Product identification studies carried out in Sub-Section 3.1.6 showed that the méthylation product was scopoletin and not its isomer isoscopoletin. It is therefore reasonably certain that scopoletin is the only product formed when aesculetin is the substrate for the 1-HMT enzyme of A. altissima cell suspension cultures. The HPLC system however separated fraxidin from its isomer isofraxidin (Table 2-15, Sub-Section 2.4.1). The méthylation product of fraxetin méthylation was identified as isofraxidin and not its isomer fraxidin by the HPLC techniques, the TLC techniques (Table 2-13, Sub-Section 2.4.1) and product identification studies carried out in Sub-Section 3.1.6.

3.2.2.1 Cell Line 1 3.2.2.1.1 Coumarin Production Results obtained from time course studies of coumarin levels and corresponding CMT activity in cell line 1 of A. altissima cell suspension cultures are presented in Figure 3.2-3. These results demonstrated that the methylated coumarins, scopoletin and isofraxidin were produced at levels of 0.04 to 0.38 mg . g dry wt. of cells ' and 0.04 to 0.35 mg . g dry wt. of cells ' respectively as shown in Figure Figure 3.2-3(a). Coumarin levels were low in comparison with canthin-6-one levels (canthin-6-one levels are a hundred fold greater than coumarin levels). The levels of scopoletin and isofraxidin produced by the cell cultures of A. altissima were similar

156 FIGURE 3.2-3 Time Course Study of Coumarin Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell line 1)

a. Coumarin levels 0 .40 - ▼ Scopoletin o Isofraxidin

0.32 -

0.24 - O)

0.16 - CO

0.08 -

0.00 - b. Coumarin methyltransferase activity using 20000 - A Aesculetin ? & □ Fraxetin /S as substrates / \ 9r 1 6000 -

12000 -

fa 8 0 0 0 -

LU 4 0 0 0 -

3 15 - c. Cell growth

(/>

14 21 28 Time (Days)

157 although peaks of activity occurred at different times in the growth cycle. Scopoletin production reached peak levels of 0.4 mg . g dry wt of cells'% early in the growth cycle, at day 12, with a smaller peak of 0.14 mg . g dry wt of cells % occurring at about day 28. Isofraxidin levels on the other hand, reached a peak level of 0.35 mg . g dry wt of cells % later in the growth cycle i.e. at day 30.

3.2.2.1.2 CMT Activity An investigation of the corresponding coumarin methyltransferase activity was subsequently carried out and the results shown in Figure 3.2-2(b). Substrates used for this investigation include aesculetin and fraxetin. Fraxetin méthylation by the CMT enzyme was found to be prominent from day 8 of the growth cycle, reaching maximum levels at about day 18. Maximal coumarin methyltransferase activity (18,000 dpm), was higher than maximal 1-HMT activity in cell line 1 (16,0(X) dpm). In spite of this fact, the canthin-6-one alkaloid méthylation product, 1 -methoxycanthin- 6-one was present at levels of 100 fold greater than fraxetin méthylation product, isofraxidin . Méthylation of aesculetin by the CMT enzyme was less prominent than fraxetin méthylation and no apparent peaks of activity occurred during the growth cycle, apart from a slight increase in aesculetin méthylation at day 12.

3.2.2.2 Cell Line 2 3.2.2.2.1 Coumarin Production Results presented in Figure 3.2-4(a), show the coumarin production pattern of this cell line. The methylated coumarins, scopoletin and isofraxidin were produced at levels of 0.02 mg . g dry wt.of cells'' and 0.04 to 0.24 mg . g dry wt. of cells ' respectively. These results show that as in cell line 1, comparable levels of scopoletin and isofraxidin were produced in cell line 2, although they had peaks of activity at different times in the growth cycle. Scopoletin production reached peak levels of 0.28 mg . g dry wt. of cells ' early in the growth cycle, at day 8, with a smaller peak of

158 FIGURE 3.2-4 Time Course Study of Coumarin Production and Corresponding Methyltransferase Activity by A. altissima Cell Suspension Cultures (Cell line 2)

0.30 - a. Coumarin levels T Scopoletin o Isofraxidin

0.24 -

0.18 - e o>

0.12 - O f)

0.06 -

0.00 - 1 5000 - b. Coumarin methyltransferase activity using A Aesculetin & □ Fraxetin ^ 12000 - as substrates

9 0 0 0 -

6 0 0 0 -

3 0 0 0 - UJ

— 10.0 - c. Cell growth

8.0 -

o 6.0 -

o> 4.0 -

(/) 2.0 - u_ 0.0 O 0 8 16 24 32 40 Time (Days)

159 0.12 mg . g dry wt of cells ’, occurring at day 28. Isofraxidin levels on the other hand reached peak levels of about 0.24 mg . g dry wt of cells'’ towards the end of the growth cycle, at day 30.

3.2.2.2.2 CMT Activity An investigation of the corresponding coumarin methyltransferase enzyme activity was carried out and the results are presented in Figure 3.2-4(b). As with cell line 1, the substrates used include aesculetin and fraxetin. In cell line 2 méthylation of aesculetin by the coumarin methyltransferase enzyme appeared to be more prominent than fraxetin méthylation. Méthylation of aesculetin was found to oscillate throughout the growth cycle, with a distinct peak of activity occurring at day 12. There were slight fluctuations in fraxetin méthylation which was also found to oscillate throughout the growth cycle, without any distinct peaks of activity.

3.2.2.2.3 Méthylation of Coumarins by the CMT Enzyme This section focuses on results obtained from studies of the méthylation of aesculetin and the méthylation of fraxetin by the CMT enzyme from A. altissima cell line 2, from cultures harvested at day 16 of the growth cycle. These experiments were carried out to investigate the following: (1) the ability of the extracted CMT enzyme from cell suspension cultures of A. altissima, to monomethylate aesculetin to scopoletin and or its isomer isoscopoletin (2) the ability of the enzyme to further methylate the monomethylated isomers, scopoletin and isoscopoletin, to yield the dimethylated coumarin scoparone (6,7- dimethoxycoumarin) as shown in Sequence 1 (Equation 3.1-101, Sub-Section 3.1.6.2). (3) the ability of the CMT enzyme from A. altissima cell suspension cultures to monomethylate fraxetin to fraxidin and or its isomer isofraxidin . (4) the ability of the enzyme to further methylate the dimethylated isomers, fraxidin and isofraxidin to yield the trimethylated coumarin, 6,7,8-trimethoxycoumarin as shown in Sequence 2 (Equation 3.1-102, Sub-Section 3.1.6.2). The results of these experiments are reported in detail in Section 3.5.

160 These results showed that in Sequence 1, méthylation of aesculetin to scopoletin by the CMT enzyme of A. altissima was significant. Méthylation of isoscopoletin also occurred at lower levels, but scopoletin méthylation was non-existent (Tables 3.5-10, Sub-Section 3.5.2). These results showed that CMT failed to methylate scopoletin which is present in cell suspension cultures of A. altissima but methylated isoscopoletin which has not been identified in A. altissima cell cultures to presumably form scoparone (6,7-dimethoxycoumarin). These results, strongly suggest that scoparone is not produced by these cultures. In Sequence 2 for fraxetin méthylation, the results obtained from preliminary experiments carried out to investigate the dimethylation of fraxetin to 6,7,8- trimethoxycoumarin (reported in detail in Table 3.5-11, Sub-Section 3.5-2), showed that fraxetin méthylation occurred to form isofraxidin . Méthylation of fraxidin also occurred, to a lower extent, but méthylation of isofraxidin was non-existent. These results showed that CMT failed to methylate isofraxidin which is present in cell cultures of A. altissima, but methylated fraxidin which has not been identified in these cultures to presumably form 6,7,8-trimethoxycoumarin. These results suggest that 6,7,8-trimethoxycoumarin is unlikely to occur in these cultures. These results were further substantiated by experiments carried out to investigate the levels of coumarins in extracts of A. altissima cell cultures (obtained as described in Sub-Section 2.2.1). This was done by employing TLC (Sub-Section 2.2.2.1 and Table 2-13, Sub-Section 2.4.1) and HPLC (Sub-Section 2.2.2.2 and Table 2-15, Sub-Section 2.4.1) techniques. Coumarins investigated include: aesculetin, scopoletin, isoscopoletin and scoparone from sequence 1, as well as fraxetin, isofraxidin and fraxidin from sequence 2 of coumarin méthylation (Sub-Sections 3.1.6.2 and 3.5.2). The level of 6,7,8-trimethoxy coumarin from sequence 2, was not investigated, due to the unavailability of the reference compound. Using these techniques, only scopoletin from sequence 1 and isofraxidin from sequence 2 of coumarin méthylation were detected in extracts from cell cultures of A. altissima. Aesculetin, isoscopoletin and scoparone, from sequence 1, as well as fraxetin and fraxidin from sequence 2 of coumarin méthylation, were not detected in extracts of these cultures.

161 3.3 An Appraisal of Potential Purification Procedures for the 1-HMT Enzyme in A. altissima Cell Suspension Cultures A series of procedures described in detail in Sub-Sections 2.3.2, 2.3.4 and 2.3.5, were proposed for the purification of the 1-HMT enzyme of A. altissima (Figure 3.3-1). The first two steps, common to all the purification procedures, involved the extraction of the crude dialysate from cell suspension cultures of A. altissima (cell line 2), harvested between days 16 and 22 of the growth cycle. This was followed by treatment of the crude dialysate with (NH4)2S0^ to obtain a precipitate at 40 % - 70 % saturation. The (NH 4)2S04 precipitate was reconstituted in buffer B(l), and the resulting enzyme solution desalted using a Pharmacia PD,o column. The enzyme solution was then purified further by using column chromatographic techniques. The chromatographic techniques employed involved the use of the following columns: DEAE cellulose anion exchange column; hydroxylapatite column; Mono-Q and Q-Sepharose anion exchange columns and Sephdex G-50, Sephacryl S-200 and Pharmacia Superose 12 HR 10/30 gel filtration columns. These column chromatographic techniques were run under conditions described in detail in Sub- Section 2.3.5. The proposed purification procedures involved the combination of two or more of these chromatographic techniques to obtain a series of purification procedures, each comprising 2 or more steps. The purification procedures are presented in Figure 3.3-1, as sequences la, 2a, 3a, 4a and 5a. Each purification procedure was assessed for efficacy and results obtained from each procedure compared in order to make a selection of the perfunctory purification procedure. The efficacy of each of these purification procedures was assessed on the basis of the following parameters, (which constitute the conventional method for assessing a purification procedure): 1. Total enzyme activity obtained after each purification step in Pkat 2. Total protein levels obtained after each purification step in mg. 3. The specific activity of the enzyme after each purification step in Pkat . g ' of protein.

162 FIGURE 3.3-1 Purification Sequences used for the Extraction and Purification of the 1-HMT Enzyme from A. altissima Cell Suspension Cultures

Extraction of harvested cells and treatment of extract with XAD-2 resin

NH2 SO4 precipitation of proteins, reconstitution of 40 % - 70 % precipitate in buffer B(1) and desalting on a Pharmacia PD^q column

Sequence la 2a Sequence 3a Sequence 4aSequence U)Os Sequence 5a DEAE cellulose Hydroxyl DEAE cellulose DEAE cellulose Sephacryl S-200 column apatite column column column column Sephadex G-50 Hydroxyl column apatite column Superose HR Mono-Q column column 4. The percentage of enzyme activity recovered after each purification step. 5. The level of purification attained after each step in the purification sequence. This is expressed as the purification fold.

These parameters were calculated for each purification sequence and results obtained were presented in a tabulated form. The Tables (Tables 3.3-10 to 3.3-14), express the efficiency of each purification sequence, on the basis of the stated parameters.

3.3.1 Purification Sequence la The chromatographic steps involved in purification sequence la are shown in sequence la of Figure 3.3-1. The reconstituted and desalted 40 % - 70 % (NH^) 2S04 precipitate was applied to a DEAE cellulose column. Figure 3.3.-2, shows the elution profile of proteins from the DEAE cellulose column. Fractions collected from this column were assayed for 1-HMT activity and CMT activity (using aesculetin as substrate) and Figure 3.3-2, shows that at this stage in the purification sequence, the 1-HMT and CMT activities may be separated. CMT activity corresponded with the first protein peak, eluted from the column with the void volume. 1-HMT on the other hand, coincided with the second protein peak (which was eluted with 200 mM KCl), indicating that this enzyme was strongly bound to the column. The most active fractions exhibiting 1-HMT activity were collected and constituted a tight band. The active 1-HMT enzyme was desalted and subsequently subjected to separation using FPLC on either a Pharmacia Mono-Q HR 5/5 column or a manually packed Q-Sepharose column. These two columns, served the same purpose in the further purification of the active 1-HMT enzyme, as relevant proteins bound to both gels in a similar manner. Figures 3.3-3 and 3.3-4, show the elution profile of proteins from the Mono-Q and Q-Sepharose columns respectively. The active 1-HMT enzyme was tightly bound to both the Mono-Q and Q- Sepharose gels and in both cases 1-HMT was eluted at a salt gradient of about 0.25 M KCl, however enzyme activity recovered from the Q-Sepharose column was

164 found to spread over a volume of 7 ml, as opposed to the Mono-Q column, where activity was confined to 1 ml. The fractions collected from the Mono-Q and Q-Sepharose columns were assayed for 1-HMT activity and a tight band of active proteins were bulked for carrying out further purification and experimentation. The Mono-Q column step appeared to be more effective than the Q-Sepharose step, as it resulted in less dilution of the enzyme preparation, however the Q- Sepharose step was easier to use because it is robust and easier to maintain than the pre-packed Mono-Q column. The final step in purification sequence la involves the use of the Pharmacia Superose 12 HR 10/30 column for further purification of the Mono-Q or Q-Sepharose column eluates. The bulked active 1-HMT enzyme fractions obtained from either the Mono-Q or Q-Sepharose columns were applied to the Superose column and eluted with running buffer B(l). The Superose gel separated the proteins on the basis of their molecular weights. The elution profile of proteins from this column (shown in Figure 3.3-5) was consistent with that of protein standards used for calibrating the column (Figure 3.4-21 of Sub-Section 3.4.4.1). Figure 3.3-5 shows that two main groups of proteins were eluted from the Superose column i.e. (the higher and lower molecular weight proteins). The higher molecular weight proteins were eluted before the lower molecular weight proteins as shown in Figure 3.4-21, Sub-Section 3.4.4.1. The fractions collected from this column were assayed for 1 -HMT activity and the active fractions are highlighted as shown in Figure 3.3-5. The fact that enzyme activity coincided with a single peak suggested the possibility that this enzyme purification procedure had yielded a single protein for the 1-HMT enzyme. The previously stated parameters for assessing enzyme purification (Section 3.3), were calculated for each purification step in purification sequence la and results compiled as shown in Table 3.3-10. Careful examination of the final step in the procedure (Figure 3.3-5), focuses on the obvious drawbacks of this purification

165 FIGURE 3.3-2 The Elution Profile of the Partially Purified A. altissima Methyltransferase Enzyme from the DEAE Cellulose Column

Active 1-HMT enzyme fractions collected Active CMT enzyme fractions collected 12500 Stepwise KCl gradient, where 1 = OmM KCl; 2 = 50mM KCl and 3 = 200mM KCl o\ *oI CN UV Absorbance at 280nm C

10.000 ■3 < I N 7,500 UJ N S sÇ 5.000

2 7,500

Volume (ml) FIGURE 3.3-3 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Mono-Q Column

Active I-I IM'I'enzyme fraciions collected Continuous KCl graclient, where 1 = OM KCl; 2 = Ü.5M KCl and 3 = IM KCl UV Absorbance at 280nm

K .1 12 5 - ■> I

Volume (ml) FIGURE 3.3-4 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Q-Sepharose Column

I X Active 1-HMT enzyme fractions collected Continuous KCl gradient, E XJCL where 1 = OM KCl and 2 = IM KCl C Os U V Absorbance at 2 8 0 n m OC >

E iS H 6

Volume (ml) FIGURE 3.3-5 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Superose 12 HR Column

Active 1-HMT enzyme fractions collected

UV Absorbance at 280nm

(X •S 0 .2 0 -

0.15 -

0.10 -

0 0 5 -

Voliimc (ml) sequence. It is apparent that there has been a carry through of low molecular weight proteins, from previous purification steps to the final step. The presence of high levels of low molecular weight impurities led to a rapid overload of the Superose column, resulting in a decreased loading capacity. The other disadvantage of this procedure is that it is fairly lengthy, involving five steps.

TABLE 3.3-10 Purification of the 1-HMT Enzyme of A. altissima Using Sequence la

Purification Total Total Specific Percentage Purification Step Activity Protein Activity Recovery (Fold) (pkat) (mg) (pkat . g ‘ (%) of Protein)

Crude 8.09 105 77.05 1 0 0 - dialysate

(NHJ^SO,

Precipitate 6 . 0 29.7 2 0 2 . 0 2 74.2 2.62 (40 % - 70 %)

DEAE 2.74 3.76 728.72 33.9 9.46 Cellulose

FPLC, Mono-Q 0.43 0 . 1 2 3583.33 5.3 46.51

FPLC, Superose 0.26 0.046 5652.17 3.2 73.36

As a result of these shortfalls in purification sequence la, alternative purification procedures were investigated for enzyme preparation.

3.3.2 Purification Sequence 2a Purification sequence 2a was proposed with two main objectives in mind. The first was; to make an attempt to shorten the purification procedure by omitting the anion exchange steps of purification sequence la (i.e. the DEAE cellulose and Mono-

170 Q column steps). The second objective was; to get rid of the low molecular weight protein impurities at an earlier stage in the purification sequence i.e. before the final stage of purification. Hydroxylapatite gel was introduced to replace the anion exchange steps used for enzyme purification in purification sequence la. The elution profile of proteins from the hydroxylapatite column was studied and the results compared with purification sequence 1 a, especially with reference to the recovery of activity and the protein levels at the end of the purification procedure. The chromatographic steps involved in purification sequence 2a are shown in Figure 3.3-1, sequence 2a. The reconstituted and desalted 40 % - 70 % (NH^jzSO^ precipitate was applied to a hydroxylapatite column. Figure 3.3-6 shows the elution profile of proteins from the column. Fractions collected from this column were assayed for 1 -HMT and CMT activities and the assays revealed that this column failed to separate the CMT and 1 -HMT enzyme activities. Previously stated parameters for assessing enzyme activity (Section 3.3), were calculated for each step in purification sequence 2a and results compiled as shown in Table 3.3-11. The results presented in Table 3.3-11, when compared with Table 3.3-10 (for purification sequence la), show that despite the inability of hydroxylapatite to separate the coumarin and alkaloid methyltransferase enzymes, it was effective in getting rid of some of the extraneous proteins. Total levels of proteins present in the partially purified enzyme solution following this purification step (which is the third step) was 2.9 mg, whilst protein levels of 3.76 mg were found to be present in the enzyme solutions after the DEAE cellulose step (the third step) in purification sequence la. As a result of these advantages obtained with the hydroxylapatite column, purification sequence 3a was designed to incorporate both the hydroxylapatite column and the DEAE cellulose column (which had the capacity to separate the CMT and the 1-HMT enzymes).

171 FIGURE 3.3-6 The Elution Profile of the Partially Purified A. altissima Methyltransferase Enzyme from the Hydroxylapatite Column

Active I-HMT enzyme fractions Active CMT enzyme fractions Stepwise NazPO^ gradient, 8000- where 1 = 20mM; 2 = 80mM and 3 = 140mM NazPO^ E UV Absorbance at 280nm T3a,

6.0004 o <

03 V) C s

18 24 30 36 42 48 54 66 72

Volume (ml) TABLE 3.3-11 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 2a

Purification Total Total Specific Percentage Purification Step Activity Protein Activity Recovery (Fold) (pkat) (mg) (pk at. g ' (%) of Protein)

Crude 11.09 196 56.6 1 0 0 - dialysate

(NHJ2 SO 4 Precipitate 7.81 27.3 286 70.4 5.05 (40-70%)

Hydroxylapatite 2 . 8 8 2.9 993 26.0 17.5

3.3.3. Purification Sequence 3a Purification sequence 3a was introduced to improve on the efficiency of purification sequence 2a by combining the previously stated advantages offered by the hydroxylapatite and DEAE cellulose columns. The chromatographic steps involved in this purification sequence are shown in Figure 3.3-1, sequence 3a. The reconstituted and desalted 40 % - 70 % (NH 4)2S04 precipitate was applied to the DEAE cellulose column (as previously described for purification sequence la in Sub-Section 3.3.1). The elution profile of proteins from the DEAE cellulose column is similar to that shown in Figure 3.3-2. The bulked active 1-HMT enzyme fractions from this column were applied to the hydroxylapatite column and the active 1-HMT fractions eluted from the hydroxylapatite column were bulked. The elution profile of proteins from the hydroxylapatite column are similar to that shown in Figure 3.3-6 of purification sequence 2a. Previously stated parameters for assessing enzyme purification (Section 3.3) were calculated for each purification step in purification sequence 3a. Results obtained from this purification sequence are presented in Table 3.3-12. These results show that, there was only a slight improvement in the loss of extraneous proteins (2

173 mg of protein was present in the enzyme following the hydroxylapatite step in purification sequence 3a and 2.9 mg following the hydroxylapatite step in purification sequence 2a). This method did not rectify the problem of the Superose column being overloaded as a result of the presence of high levels of extraneous low molecular weight proteins. Protein levels of 2 mg in the hydroxylapatite column resulted in an overload of the Superose column just as 0.12 mg of protein in the Mono-Q eluate did. This purification procedure failed to accomplish the purpose for which it was designed, therefore purification sequence 4a was introduced.

TABLE 3.3-12 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 3a

Purification Total Total Specific Percentage Purification Step Activity Protein Activity Recovery (Fold) (pkat) (mg) (pk at. g ’ (%) of Protein)

Crude 9.3 189 49.2 100 - dialysate

(NHASO, 6.3 19 331.6 67.8 6.7 Precipitate (40 % - 70 %)

DEAE 1.91 3.3 578.8 20.5 11.8 Cellulose

Hydroxyl 1.88 2.0 940 20.2 19.1 apatite

3.3.4 Purification Sequence 4a Purification sequence 4a for the 1-HMT enzyme was devised with emphasis on a different approach from the previous purification sequences. The emphasis was on the early introduction of a gel filtration step into the procedure used in purification sequence la. The gel filtration column is expected to remove low molecular weight

174 extraneous proteins, from the enzyme preparation, before the application of the final purification step. The chromatographic steps involved in this purification sequence are shown in Figure 3.3-1, sequence 4a. The reconstituted and desalted (NH^) 2S04 precipitate was applied to a DEAE cellulose collum and the active 1-HMT enzyme fractions collected, were applied to a Sephadex G-50 column (a gel filtration column which had the capacity to separate globular proteins of molecular weight ranging from 1,500 to 30,000 Dalton). Figure 3.3-7, shows the elution profile of proteins from the Sephadex G-50 column. The active fractions from this column were bulked for carrying out enzyme assays and the determination of levels of total protein. The Sephadex G-50 column resulted in an obvious dilution of the active fractions to a volume of about 22 ml as compared to the volume of 8 ml applied to it. The levels of total protein present in the combined active fractions following the Sephadex G-50 step (3.6 mg) was still quite substantial. A subsequent Mono-Q step was therefore introduced to purification sequence 4a. This column has the capacity to concentrate large volumes of the active proteins to much smaller volumes and also to further remove some of the extraneous proteins. This step involved the application of the active Sephadex G-50 eluate to a Mono-Q column. This purification step resulted in a purification profile similar to that shown in Figure 3.3-3 from sequence la. This step reduced protein levels to 0.13 mg. This procedure however still resulted in an overload of the Superose column. The previously mentioned parameters for assessing enzyme purification (Section 3.3), were calculated for each step in this purification sequence and results presented in Table 3.3-13. These results showed that the total protein present in the active fractions at the end of the final purification step (0.13 mg) still contained comparable levels of extraneous proteins to previous purification procedures. This procedure was therefore not successful in the removal of sufficient levels of low molecular weight extraneous proteins, even though the elution pattern of proteins from the superose column in purification sequence la (Figure 3.3-5) had suggested that a Sephadex G-50 column would be ideal for the removal of low molecular weight proteins. (Sephadex G-50 gel has the capacity for separating globular proteins of

175 FIGURE 3.3-7 The Elution Profile of the Partially Purified 1-HMT Enzyme of A. altissima Cell Suspension Cultures from the Sephadex G-50 Column

1800

1650

1500 Active 1-HMT enzyme fractions collected

UV Absorbance at 280nm 1350 o\ g 1200 f 1050

900 o CO u 750 ■ E N 600 - W H 450 ■

5 300 -

150 -

Volume (ml) molecular weight ranging from 1,500 to 30,000).

TABLE 3.3-13 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 4a

Purification Total Total Specific Percentage Purification Step Activity Protein Activity Recovery (Fold) (pkat) (mg) (pk at. g ‘ (%) of Protein)

Crude 10.12 178 56.9 100 - dialysate

(NHJ2 SO 4 7.3 39 187.2 72.1 3.3 Precipitate (40 % - 70 %)

DEAE 3.06 4.13 740.9 30.2 13.0 Cellulose

Sephadex G-50 2.59 3.6 719.4 25.6 12.6

Mono-Q 0.58 0.13 4461.5 5.7 78.4

The short comings of this purification procedure appear to have arisen mainly from the Sephadex G-50 gel filtration step, which did not fulfil the desired expectations. It was therefore reasoned that this procedure could be improved upon by replacing the Sephadex G-50 step with a different gel filtration step i.e. the Sephacryl S-200. It was also reasoned that as a result of the length of time required to run an average gel filtration column, it was necessary to shorten the purification procedure by omitting one of the previous purification steps. This led to the introduction of purification sequence 5a.

3.3.5 Purification Sequence 5a Purification sequence 5a, was similar to sequence 4a, except for the replacement of the Sephadex G-50 column step with the Sephacryl S-200 column step.

177 and the exclusion of the DEAE-Cellulose step in an attempt to shorten the purification procedure. The Sephacryl S-200 gel filtration column was selected because it has a much larger capacity than the Sephadex G-50 column and was therefore expected to be more efficient. The Sephacryl S-200 gel filtration column has the ability to separate globular proteins of molecular weight ranging from 5000 to 250,000. The chromatographic steps involved in purification sequence 5a are shown in Figure 3.3-1, sequence 5a. The reconstituted and desalted 40 % - 70 % (NH 4)2S04 precipitate was applied to a Sephacryl S-200 column. Figure 3.3-8, shows the elution profile of proteins from the Sephacryl S-200 column. The previously stated parameters for assessing enzyme purification (stated in Section 3.3), were calculated for each step in this purification sequence and results presented in Table 3.3-14. These results showed that the Sephacryl S-200 step was even less efficient than the Sephadex G-50 step (in purification sequence 4a) in the removal of extraneous proteins. Protein levels following the Sephacryl S-200 step in purification sequence 5a (17.37 mg) were much higher than levels obtained after the Sephadex G-50 step in purification sequence 4a (3.6 mg). This is because the DEAE cellulose step used in sequence 4a was omitted in sequence 5a. The Sephacryl S-200 column like the Sephadex G-50 column also resulted in the dilution of the sample to a volume of 22 ml as compared to the volume of 6 ml applied to it. This resulted in a dilution of specific enzyme activity to very low levels of 272 Pkat. g ’ of protein. The Sephacryl S-200 eluate was therefore applied to a Mono-Q column (for concentration of enzyme activity and for further removal of extraneous proteins). The elution profile of proteins from this column is similar to that shown in Figure 3.3-3 from sequence la. Protein levels of 0.11 mg obtained after the Mono-Q column step also resulted in an overload of the superose column.

178 FIGURE 3.3-8 The Elution Profile of the Partially Purified A. altissima Methyltransferase Enzyme from the Sephacryl S-200 Column

Active 1-HMT enzyme fractions Active CMT enzyme fractions

1200 UV Absorbance at 280nm

1000

VO 800 -

600 -

200 -

Volume (ml) TABLE 3.3-14 Purification of the 1-HMT Enzyme of A. altissima Using Sequence 5a

Purification Total Total Specific Percentage Purification Step Activity Protein Activity Recovery (Fold) (pkat) (mg) (pk at. g ' (%) of Protein)

Crude 8.44 145 58.2 100 - dialysate

(NHJzSO, Precipitate 6.9 21.54 320 81.8 5.5 (40 % - 70 %)

Sephacryl S-200 4.72 17.37 272 55.9 4.7

Mono-Q 0.48 0.11 4364 5.7 75

3.3.6 The Choice of a Perfunctory Purification Procedure The procedure for routine use in the purification of the 1-HMT enzyme of A. altissima cell suspension cultures was selected from the five tested procedures (purification sequences la to 5a described in Sub-Sections 3.3-1 to 3.3-5). An assessment of the efficiencies of these procedures was made on the basis of the parameters for assessing enzyme purification (Section 3.3). These parameters were determined for each of the procedures and tabulated as shown in Tables 3.3-10 to 3.3- 14. The factors taken into consideration in making the final choice were the speed, convenience, and efficiency of purification. Purification sequence la was selected as the procedure for the perfunctory purification of 1-HMT. Purification sequence la (results presented in Table 3.3-10), is a five step procedure which is quite efficient with respect to the purification capacity it exhibited. This is reflected by the relatively high purification fold obtained at the penultimate and final steps of the purification procedure (46.51 and 73.36 fold respectively). This was also complemented by a high specific activity of the proteins

180 at the penultimate and final steps of the purification (3583.33 and 5652.17 Pkat . g ' of protein respectively). Another advantage exhibited by this purification procedure is the time required to run the procedure. Even though it is a five step procedure, the time required for running purification sequence 1 a, is considered to be comparatively short since all the individual steps of this procedure with the exception of the final Superose step are quite rapid. This purification procedure had 2 disadvantages. The first disadvantage is the percentage recovery of proteins, which was found to be relatively low (5.3 %). This was indicative of the loss of a fair amount of the active protein during the purification. The major drawback however is the fact that it resulted in an overload of the Superose column, thereby decreasing the amount of partially purified enzyme that could conveniently be loaded on this column at any time. In spite of this shortfalls, purification sequence la was eventually selected as the purification procedure for routine use as it appeared to have the least significant disadvantages. The other procedures were eliminated due to reasons which will subsequently be discussed. Purification sequence 2a (Table 3.3-11), is a rapid three step procedure which was eliminated because it was unable to get rid of sufficient levels of extraneous proteins. Furthermore the hydroxylapatite column did not have the capacity to separate the coumarin and alkaloid methyltransferases. Purification sequence 3a (Table 3.3-12), a four step procedure was able to eliminate the coumarin methyltransferase enzyme as a result of the incorporation of the DEAE cellulose step into sequence 2a. Purification sequence 3a was therefore a little more efficient than sequence 2a for the removal of extraneous proteins, but still not efficient enough to provide a significant improvement over sequence la. Purification sequence 4a (Table 3.3-13) is a five step procedure, which proved to be of similar efficiency to sequence la, resulting in a fairly high level of purification of the 1-HMT enzyme (78.5 fold). It also produced a relatively high specific activity of 4461.5 Pkat . g ‘ of protein, following the Mono-Q column step. This procedure also exhibited similar efficiency to sequence lain the removal of

181 extraneous proteins (protein levels following the Mono-Q column step was 0.13 mg in sequence 4a and 0.12 mg in sequence 1 a). However in order to purify the enzyme to a single protein as was the case in purification sequence la, a final superose gel filtration step would have to be incorporated into this procedure. On this basis, purification sequence 4a was bypassed in favour of the almost equally efficient purification sequence la, which is one step shorter. Purification sequence 5a (Table 3.3-14) is a five step procedure which is similar to sequence 4a. This procedure was intended to be an improvement over sequence 4a (with respect to the elimination of extraneous proteins and the running time), by excluding the DEAE cellulose step and replacing the sephadex G-50 step in purification sequence 4a with a Sephacryl S-2(X) step. This procedure demonstrated a similar efficiency to sequence 4a (it had a relatively high specific purification capacity as reflected by the purification fold, 75 fold), as well as a high specific activity of 4364 P kat. g ' of protein, after the last purification step. However as with sequence 4a, a final Superose gel filtration step would have to be incorporated, for purification to a single protein to occur. Even though there was a slight improvement in running time over sequence 4a due to the exclusion of the DEAE cellulose step, purification sequence 5a still remained a lengthy one requiring two gel filtration steps i.e. the Sephacryl S-200 and Pharmacia Superose steps. Purification sequence 5a offered no advantage over purification sequence 4a in getting rid of extraneous proteins as the Mono-Q eluate still contained comparable levels of extraneous proteins to previous purification procedures. As a result of these disadvantages, this procedure was eliminated for perfunctory use.

182 3.4 Characterisation of the 1-HMT Enzyme of A. altissima Cell Suspension Cultures Characterisation of this enzyme involved the following: determination of conditions required for optimum enzyme activity; kinetic properties of the enzyme substrates; inhibitor studies and the determination of inhibition constants, molecular weight determination; stability studies and substrate specificity studies. Experiments involving the characterisation of the 1-HMT enzyme were performed on the enzyme extracted from cell line 2 of A. altissima cell suspension cultures harvested between days 16 to 22 of the growth cycle.

3.4.1 Optimum Conditions for Enzyme Activity The conditions required for optimum 1-HMT activity, were determined by carrying out assays for enzyme activity at two different levels of enzyme purification. This was done for comparative purposes, in order to investigate the effect of the level of enzyme purification on the conditions required for optimal enzyme activity. The enzyme preparations employed in these studies are the DEAE cellulose and the Mono- Q eluates from purification sequence la (Sub-Section 3.3-1). These enzyme preparations were also used in subsequent experiments involving the characterisation of the enzyme. The conditions investigated for optimal enzyme activity include; optimum protein levels, incubation time, temperature and pH. The protein levels and incubation time have a general applicability to enzyme catalysed reactions. Early studies of enzyme catalysed reactions revealed that the rate of an enzyme catalysed reaction is directly related to the concentration of the enzyme in the presence of saturating substrate levels. It was therefore of great importance to determine the optimum protein levels of the enzyme. The catalytic activity of enzymes is also very sensitive to temperature and pH, with deactivation common at both high and low extremes of each parameter. It was thus necessary to determine the optimum temperature and pH associated with maximum activity. Each of the conditions stated above were individually investigated for their

183 effect on enzyme activity. Experiments involved carrying out enzyme assays on standard incubation mixes (Sub-Section 2.3.3.1), which were incubated under varying conditions. During the study of each condition, one condition was varied whilst the others were kept constant. Enzyme assays were performed in triplicate on standard incubation mixes, which were incubated under pre determined optimum conditions for enzyme activity. Enzyme activity in dpm was then determined as described in Sub-Section 2.3.3.3 .

3.4.1.1 Optimum Protein Levels The protein levels required for optimum enzyme activity were determined for each of the two levels of enzyme purification investigated i.e. the DEAE cellulose and Mono-Q eluates from purification sequence la (Sub-Section 3.3.1). Volumes of enzyme solution (from each of these purification levels), corresponding to levels of protein ranging from 15 to 100 pg, for the DEAE cellulose eluate and 6 to 50 pg for the Mono-Q eluate were individually incubated in the standard incubation mix. The incubation mixes were then assayed for enzyme activity as described in Sub-Section 2.3.3. Graphs of enzyme activity versus protein levels in pg were plotted for each of the enzyme purification levels investigated as shown in Figures 3.4-1 and 3.4-2 respectively. The rate of an enzyme catalysed reaction is directly related to the concentration of the enzyme and is therefore expected to increase with increasing enzyme concentration, when substrates are present in saturating levels. These results shown in Figures 3.4-1 and 3.4-2 revealed that enzyme activity was directly proportional to protein levels for the DEAE cellulose eluate (Figure 3.4-1) from protein levels of 15 to 49 pg and for the Mono-Q eluate (Figure 3.4-2), from protein levels of 6 - 24 pg.

184 FIGURE 3.4-1 The Effect of Protein Levels on 1-HMT Activity (1)

1500 -

S - 1 200 - Optimum Protein Levels: 15-49 jjg

900 -

600 -

LU 300 -

0 20 40 60 80 100

Protein Levels (pg)

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

185 FIGURE 3.4-2 The Effect of Protein Levels on 1-HMT Activity (2)

10000 -

E ^ 8000 - Optimum Protein Levels: 6 -2 4 pg

> 6000 - o < 0 ) 4000 - E >* N c LU 2000 -

0 10 20 30 40 50

Protein Levels (pg)

The assay was done under standard conditions using active fractions from the Mono-Q column in purification sequence 1

186 3.4.1.2 Optimum Incubation Time The incubation time required for optimum enzyme activity was investigated at two different stages of enzyme purification i.e the DEAE cellulose and Mono-Q eluates from purification sequence la (Sub-Section 3.3-1). Samples of enzyme solution from each of these purification stages were assayed for activity, following incubation in the standard incubation mixes for periods of time ranging from 15 to 90 minutes. Results of enzyme activity versus incubation time were plotted for each of the two enzyme purification levels investigated i.e. the DEAE cellulose and Mono-Q eluates, as shown in Figures 3.4-3 and 3.4-4 respectively. With the aid of these graphs it was possible to select the optimum incubation time at each purification stage. The optimum incubation time was determined as the incubation time, where there was maximal enzyme activity within the linear region of the graph. Optimum incubation time was found to be 45 minutes for both the DEAE cellulose and Mono-Q eluates as shown in Figures 3.4-3 and Figure 3.4-4 respectively. In both cases however there was a linear relationship between enzyme activity and incubation time for periods of time ranging from 15 to 45 minutes. It is also apparent by comparing Figures 3.4-3 and 3.4-4 that optimum incubation time did not vary with progressive purification of the enzyme.

3.4.1.3 Optimum Temperature The optimum temperature for enzyme activity was investigated at 2 different stages of purification i.e the DEAE cellulose and Mono-Q eluates from purification sequence la. This was achieved by incubating the partially purified enzyme at temperatures ranging from 20 to 50 °C, at intervals of 5 °C. 30 minute Incubations were carried out in standard incubation mixes, which were subsequently assayed for enzyme activity, at each of the chosen temperatures. Results of enzyme activity versus temperature were plotted for both the DEAE cellulose and Mono-Q eluates, as shown in Figures 3.4-5 and 3.4-6 respectively. These graphs were used to determine the optimum temperature, which was considered as the temperature at which

187 FIGURE 3.4-3 The Effect of Incubation Time on 1-HMT Activity (1)

15000 -

E 12000 - T3 Optimum Incubation Time: 15-45 minutes.

9000 - o (O 0 ) 6000 - E >. N iS 3000 -

0 18 36 54 72 90

Incubation Time (minutes)

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

188 FIGURE 3.4-4 The Effect of Incubation Time on 1-HMT Activity (2)

2000 -

B- 1600 - Optimum Incubation Time: 15-45 minutes.

1200 -

800 -

LU 400 -

0 12 24 36 48 60

Incubation Time (minutes)

The assay was done under standard Conditions using active fractions from the Mono-Q column in purification sequence 1

189 FIGURE 3.4-5 The Effect of Temperature on 1-HMT Activity (1)

25000 -

E Q. ■O 20000 - Optimum Temperature: 35 *0

15000 - O <

• N C LJJ 5000 -

Temperature (*C)

The assay was done under standard conditions, with an incubation time of 30 minutes, using active fractions from the DEAE-Cellulose column in purification sequence 1 .

190 FIGURE 3.4-6 The Effect of Temperature on 1-HMT Activity (2)

30000 -

E ^ 24000 - Optimum Temperature: 35 "C

18000 - u (Ü Q) 12000 - E >* N iM 6000 -

14 20 26 32 38 44

Temperature (*C)

The assay was done under standard conditions, with an incubation time of 30 minutes, using active fractions from the Mono-Q

column in purification sequence 1 .

191 maximum activity occurred. The optimum temperature of 1-HMT did not appear to be affected by the level of purity of the enzyme as the optimum temperature for both the DEAE cellulose and the Mono-Q elutes is 35 °C, as shown in Figures 3.4-5 and 3.4-6 respectively. Temperatures above 35 °C were deleterious to enzyme activity. Although the optimum temperature is 35 °C, temperatures of 30 °C were employed for standard assays because at longer incubation periods, a lower degree of dénaturation is expected to occur at lower temperatures.

3.4.1.4 Activation Energy Any chemical reaction represents a transition from one state (reactants) to another (products). However the progress of the reaction, energetically speaking does not proceed directly from reactants to products. Modem kinetic theory proposes that product formation proceeds via the formation of a transition state, corresponding to an activated (higher energy) state of the reactants. The velocity of the conversion is consequently governed by the ease with which the transition state is achieved. In the context of the transition state theory, a catalyst functions by enhancing the production of the transition state i.e it reduces the energy of activation without affecting the net energetics of the overall reaction (Bohinski, 1979 a). The activation energy of the proteins present in the DEAE cellulose and the Mono-Q eluates from purification sequence la were determined in order to investigate the catalytic efficiencies of both enzyme preparations and to compare the effect of relative purity of the enzyme preparation on its catalytic efficiency. Activation energy determination was achieved by employing the Arrhenius equation which relates the specific rate constant to temperature (Bohinski, 1979 a). This was done individually for both the DEAE cellulose and the Mono-Q enzymes. The experimental procedure involved carrying out enzyme assays on standard incubation mixes, following incubation, at temperatures of 20, 25, 30 and 35 °C for a period of 30 minutes. These temperatures are considered as temperatures at which the enzyme has the ability to function at its full capacity, without the hindrance of thermal dénaturation. Results for experiments carried out with both the DEAE cellulose and Mono-Q

192 FIGURE 3.4-7 The Arrhenius Plot for the 1-HMT Enzyme (1)

q d E Q. ~o CO o> >» 4—«

’> C\J o o> E. = 70.8 kJ.M < a> 00 E CO >• N C 111 CO

o 00

0.0030 0.0031 0.0032 0.0033 0.0034 0.0035

Temperature"’-1 (*K)

The assay was done under standard conditions using the active fractions from the DEAE-Cellulose column in purification sequence 1

193 FIGURE 3.4-8 The Arrhenius Plot for the 1-HMT Enzyme (2)

10.4

9.6 - > 4-^ o E. = 84.4 kJ.M“ < 9.2 - 0) E >* N 8.8 - C LU 8.4 - c _J

0.0030 0.0031 0.0032 0.0033 0.0034 0.0035

Temperature"' (*K)

The assay was done under standard conditions using active fractions from the M ono-Q column in purification sequence 1

194 enzyme preparations were plotted as shown in Figures 3.4-7 and 3.4-8 respectively. The Activation energies were subsequently determined, for the DEAE cellulose and Mono-Q enzyme preparations as 70.8 KJ.M ' and 84.4 KJ.M ' respectively, as shown in Figures 3.4-7 and 3.4-8 respectively. Comparison of the activation energies of the DEAE cellulose enzyme preparation and the purer Mono-Q preparation, shows that the enzymes have similar activation energies, indicating that the enzyme activities in both cases have similar efficiencies, despite the presence of other extraneous proteins.

3.4.1.5 Optimum pH The optimum pH for enzyme activity was determined at two different levels of enzyme purification, the DEAE and Mono-Q eluates from purification sequence la. Each of the enzyme solutions was incubated at pH levels ranging from 5.6 to 9 using three different buffer systems to buffer the enzyme solutions to the required pH. The buffers used were previously described in Sub-Section 2.3.1. Buffers of the C(l) range i.e. C(l)-1 and C(l)-2 are 400 mM citric acid : sodium citrate buffer, used for buffering the enzyme solutions to pH 5.6 and 6 respectively. Buffers of the C(2) range i.e. C(2)-l to C(2)-5 are 400 mM sodium phosphate buffer, for buffering the enzyme solutions to pH 6, 6.6, 7, 7.5 and 8 respectively and finally the C(3) range i.e. C(3)-l, C(3)-2 and C(3)-3 are 400 mM Tris-HCl buffer for buffering the enzyme solution to pH levels of 8, 8.5 and 9 respectively. This was followed by incubation of the enzyme solution in a standard incubation mix, and the subsequent assay for enzyme activity at each pH level. The overlap of pH with every change in buffer was done to investigate the effect of buffers on pH. Results of enzyme activity versus pH of the protein solution, were plotted for both enzyme preparations investigated i.e. the DEAE cellulose and Mono-Q eluates as shown in Figures 3.4-9 and 3.4-10 respectively. These results were used for the determination of optimum pH. Since enzymes are proteins, pH changes will profoundly affect the ionic character of the amino acid and carboxylic acid groups on the protein and would therefore markedly affect the catalytic site and conformation of an enzyme. In addition to these ionic effects, low and high pH values can cause

195 FIGURE 3.4-9 The Effect of pH on 1-HMT Activity (1)

_L 5000 - ■ Buffer C(1) e Buffer C(2) E ♦ Buffer C{3) ^ 4000 Optimum >• 4—» pH : 7-7.5 ’> 3000 - 4-^ O <

0) 2000 - E N C LU 1000 -

5.0 5.8 6.6 7.4 8.2 9.0

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1. The buffers used for the determinations include: C(1)-citric acidisodium citrate buffer, C(2)-sodium phosphate buffer and C(3)-Tris-HCI buffer

196 FIGURE 3.4-10 The Effect of pH on 1-HMT Activity (2)

8000 - ■ Buffer C(1) • Buffer C(2) E ♦ Buffer C(3) CL 7100 - "O Optimum >• pH: 7-7.5 4—» ’>= 6200 - o < 5300 - m E >* N 440 0 - C LU

3500 -

5.9 6.8 7.6 8.5 9.4

The assay was done under standard conditions using active fractions from the Mono-Q column in purification sequence 1. The buffers used for the determinations include: C(1)-citric acid:sodium citrate buffer, C(2)-sodium phosphate buffer and C(3)-Tris-HCI buffer.

197 considerable dénaturation and hence inactivation of the enzyme protein (Bohinski, 1979 a). These effects seen at lower pH levels of 5.6 and 6 and higher pH levels of 8, 8.5 and 9 in both Figures 3.4-9 and 3.4-10, are probably the main determinants of a typical enzyme activity - pH relation. Thus a bell-shaped curve obtains, with a relatively small plateau and sharply decreasing rates on either side as indicated in Figures 3.4-9 and 3.4-10. The pH optima of both the DEAE cellulose and the purer Mono-Q enzyme preparations fell within a plateau range of 7 to 7.5, indicating that the pH optimum was not affected by the presence of extraneous proteins. It was also observed from results presented in Figures 3.4-9 and 3.4-10, that in both cases the sodium phosphate buffer produced significantly lower enzyme activity than using the citric acid buffer of the same pH (i.e. pH 6). The differences in activities with Tris and Sodium phosphate at pH 8 were much lower and did not appear to be significant.

3.4.2 Michaelis-Menten constants (K„) for enzyme substrates Michaelis and Menten (1913), pioneered the kinetic study of enzyme catalysed reactions and the development of theories governing enzyme participation in these reactions. The general theory proposed has since guided enzyme kinetics, although it has been elaborated upon in several ways. Early studies of enzyme catalysed reactions revealed that when the saturating substrate concentration was held constant and the amount of enzyme varied, a linear increase in velocity was observed with increasing enzyme levels as shown in Figures 3.4-1 and 3.4-2. However in experiments of the reverse type, in which the enzyme concentration was held constant and the amount of substrate varied, a non-linear hyperbolic relationship between velocity and substrate concentration was observed as shown in Figures, 3.4-12, 3.4-13 and 3.4-14. Both of these relationships have been found to have a general applicability to enzyme catalysed reactions. Michaelis and Menten reasoned correctly that an enzyme catalysed reaction at varying substrate concentrations is diphasic. At low substrate concentrations, the active sites on the enzyme molecules are not saturated by substrate and thus the enzyme reaction rate

198 varies with the substrate concentration. As the number of substrate molecules increases however, the sites are covered to a greater degree until at saturation, no more sites are available, the enzyme is working in full capacity and the reaction rate is independent of substrate concentration. Michaelis and Menten (1913), defined the fundamental quantity of enzyme kinetics, as the dissociation constant of the enzyme - substrate complex (ES) formed from the enzyme E and the substrate S.

[E] [S] Ks = ...... Equation 3.4-1(X) [ES]

Equation 3.4-100 is an expression of the rate of the reaction in Equation 3.4-101.

K, K3

E + S ES -> E + P ..... Equation 3.4-101 Kz where K^, K2 and K3 are specific rate constants for each reaction.

Briggs and Haldane (1925), extended this theory to obtain the steady state derivation (Equation 3.4-102) for enzyme catalysed reactions involving one-substrate or multisubstrate reactions where all the other substrate concentrations are held constant. This derivation which was extended to form Equation 3.4-103 was made on the assumption that: 1) there is only one kinetically active intermediate complex; 2) that K3 is very slow in comparison to K2; 3) and 4) that the total enzyme involved in the reaction [E]<, = [E] + [ES]

Kz [E] [S]

Kqj = =...... Equation 3.4-102 K, [ES]

199 is the Michaelis-Menten constant. The represents the amount of substrate required to bind with half of the total amount of enzyme present in solution. This condition is termed half saturation and corresponds to the substrate concentration at which the reaction rate has half its maximal value i.e. V2 (V^ax)- From Equation 3.4-102, the reaction rate was determined as:

V^ m ax rsi V ^ m ax V = = Equation 3.4-103 [S] + IL 1 + K^/[S]

The most commonly used method for the determination of values is from an equation derived by Lineweaver and Burk (1934). This equation is the reciprocal form of Equation 3.4-103.

1 1 1 Equation 3.4-104

Vmax [S]

The Equation corresponds to that of a straight line i.e. y = mx + c. If 1/V is plotted against 1/[S], a straight line is obtained. The gradient of the straight line = the intercept on the ordinate = 1 / and the intercept on the abscissa = c / m = -l /K^, (Michal, 1978; 1983). The factors which determine the reaction rate of an enzyme catalysed reaction are influenced by these two kinetic parameters (K^ and V^ax)- It is believed that reflects the rate at which the enzyme-substrate complex is formed. The is an expression of the efficiency of enzyme operation and reflects the rate at which the enzyme-substrate complex breaks down to form the product (Bohinski, 1979 a). The Vmax / ratio also gives an indication of the affinity of the enzyme for the substrates

200 (Ebel et a l, 1972). Substrates exhibiting low values and high values have been reported to exhibit the highest affinity for the enzyme and are therefore the best substrates (Poulton, 1981). It is therefore important to determine the and as accurately as possible (Bohinsky, 1979 a). The values of the two substrates of the 1-HMT enzyme; SAM and 1- hydroxycanthin-6-one, were determined. In addition to this, the of each of the enzyme catalysed reactions, studied during the determinations, were also calculated. These were achieved, by employing partially purified enzyme preparations from two different stages of enzyme purification i.e. the DEAE cellulose and Mono-Q eluates from purification sequence la.

3.4.2.1 K„ for SAM The Km for SAM was determined at two stages of enzyme purification (the DEAE cellulose and Mono-Q eluates from purification sequence la), by carrying out assays for methyltransferase activity in standard incubation mixes containing levels of SAM ranging from 2.1 x 10^ mM to 1 mM. The standard incubation mixes used for the enzyme assays contained the following: (1) SAM at the required concentrations, present as mixtures of radiolabelled (hot) and non-radiolabelled (cold) SAM, with hot SAM ratios ranging from 2 % to 40 %; (2) l-hydroxycanthin-6-one at the saturating level of 230 pM; and (3) 100 pi of the DEAE cellulose enzyme preparation, containing an optimum protein level of about 30 pg. Methods used for the preparation of solutions of the substrates are described in Sub-Section 2.3.3.1 . Assays for enzyme activity in each incubation mix were carried out in triplicate, under optimum conditions for activity i.e. a temperature of 30 °C, a pH of 7.5 and an incubation time of 45 minutes. The enzyme activities in pkat, corresponding to each of the SAM concentration levels investigated, were subsequently calculated as described in Sub-Section 2.3.3.4 and a Lineweaver-Burk double reciprocal plot of the reciprocal of enzyme activity versus the reciprocal of the milli-molar SAM concentration was plotted as shown in Figure 3.4-11. The as well as the were subsequently computed. The value for SAM, using the DEAE cellulose

201 FIGURE 3.4-11 A Lineweaver-Burk Double Reciprocal Plot for the Determination of the Km of SAM

15.0

I 4-» 13.6 <0

Ql >* 12.2 max

4-* O 10.8 < 0) E >• 9.4 N C LU 8.0

0 4 8 12 16 20

[SAM mM]-1

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

202 FIGURE 3.4-12 The Effect of SAM Concentration on 1-HMT Activity

0.150 -

Q l

15.0 -1 0.122 - >

K. - 11.4 - 0.108 - - 0.14 «.« -

N 0.094 - Ui c «,0 - LU 20 30 40 so

[SAM m M F ’ 0.080 -

0.00 0.16 0.32 0.48 0.64 0.80

[SAM mM]

The insert shows a Lineweaver-Burk double reciprocal plot. The assay was done under standard conditions using active fractions from the Mono-Q column in purification sequence 1

2Ü3 enzyme preparation is 24 pM and the is 0.11. The experiment was then repeated for the Mono-Q enzyme preparation by following the same procedure, but replacing the DEAE cellulose eluate with the Mono-Q eluate at a saturating protein level of about 15 pg in 100 pi. A Michaelis- Menten plot of enzyme activity versus milli-molar SAM concentration and a Lineweaver-Burk graph of the reciprocal of enzyme activity versus the reciprocal of milli-molar SAM concentrations were plotted as shown in Figure 3.4-12. The value for SAM, using the Mono-Q enzyme was subsequently computed as 18 pM and the as 0.14. Comparison of SAM values, for both the DEAE cellulose and Mono-Q eluates (Figures 3.4-11 and 3.4-12 respectively), revealed that the value decreased slightly with enzyme purity i.e. from 24 pM for the DEAE cellulose to 18 pM for the Mono-Q enzymes whilst the increased slightly from 0.11 to 0.14. It is uncertain if these differences are significant however, these results indicate that there is a slightly greater efficiency in the formation and break down of the enzyme-substrate complex, with the purer Mono-Q enzyme preparation, than with the DEAE cellulose enzyme preparation.

3.4.2 2 for l-Hydroxycanthin-6-one The of l-hydroxycanthin-6-one was determined at two different stages of enzyme purification (the DEAE cellulose and the Mono-Q eluates from purification sequence la), by carrying out assays for methyltransferase activity on standard incubation mixes containing levels of 1-hydroxycanthin 6-one ranging from 7 x 10 ^ to 7 mM. The standard incubation mixes used for the enzyme assays contained the following: l-hydroxycanthin-6-one at the required concentration; SAM at the saturating level of 60 pM, present as a hot ("^C-labelled) and cold (non-labelled) mixture with a hot ratio of 4.65 % and 30 pg of protein in 100 pi of the DEAE cellulose eluate or 15pg of protein in 100 pi of the Mono-Q eluate. The enzyme activities in pKat were calculated as described in Sub-Section 2.3.3.4, at each of the concentration levels of 1 -hydroxycanthin-6-one investigated.

204 FIGURE 3.4-13 A Lineweaver-Burk Double Reciprocal Plot for the Determination of

the Km of 1 -Hydroxycanthin- 6 -o n e

U 14 - (O Q. >• 4-» >

0) E >* N C LU

10 -

0.0 0.3 0.6 0.9 1.2 1.5

[1-Hydroxy-C-6-one mM] -1

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

205 FIGURE 3.4-14 The Effect of 1 -Hydroxycanthin-6-one Concentration on 1-HMT Activity

0.25 -

4—» <0 Q.

.ti: 0 .1 9 - 8 .0 -1 >

0) w - E N C 4J3 - LU

(1-Hydroity-C-6-on« mM]' 0.10 -

0.0 0.3 0.6 0.9 1.2 1.5

[1 “ Hydroxy—C—6—one mM]

The insert shows a Lineweaver-Burk double reciprocal plot. The assay was done under standard conditions using active fractions from the Mono-Q column in purification sequence 1

2Ü6 A Lineweaver-Burk double reciprocal plot of the reciprocal of enzyme activity versus the reciprocal of mM l-hydroxy-C-6-one concentration was plotted for the DEAE cellulose eluate (Figure 3.4-13). A Michaelis-Menten plot of enzyme activity versus mM concentrations of 1-hydroxy-C-6-one as well as a lineweaver-Burk double reciprocal plot of the reciprocal of enzyme activity versus the reciprocal of the mM concentrations of 1-hydroxy-C-6-one, were plotted for the Mono-Q eluate (Figure 3.4- 14). The (K^) for l-hydroxy-C-6-one as well as the were then computed for both the DEAE cellulose and Mono-Q eluates from purification sequence la. The value for l-hydroxy-C-6-one, using the DEAE cellulose enzyme preparation is 53.3 pM and the is 0.1. The value for 1-hydroxy-C-6-one, with the Mono-Q enzyme was computed as 22 pM and the as 0.21. The Km value obtained from the purer Mono-Q preparation was lower than the DEAE cellulose preparation whilst the V^ax was higher. Although it is uncertain if these differences are significant, these results indicate that the Mono-Q enzyme has a slightly more efficient reaction path and thus a greater ease of formation and breakdown of the enzyme-substrate complex than the DEAE cellulose enzyme.

3.4.3 Inhibitor Studies A wide range of compounds have the ability to combine with certain enzymes in either a reversible or irreversible manner thereby blocking the catalytic activity of that enzyme. Such compounds are called inhibitors and include drugs, antibiotics, poisons, antimetabolites as well as products of the enzyme reaction. Three general classes of reversible inhibitors are recognised and their actions involve competitive, non-competitive or un competitive inhibition. Optimum activity of some enzymes require the participation of a metal co-factor. If present, the metal ion functions as an activator, whilst its absence would inhibit enzyme activity. In addition to being a naturally occurring phenomenon, the inhibition of enzymes is also important in that it provides information helpful in understanding how an enzyme operates, in identifying amino acid residues essential for catalytic activity and in further clarifying issues of any specificity of action (Bohinski, 1979 a). The following section focuses on preliminary inhibitor studies of the 1-HMT

207 enzyme using the DEAE cellulose eluate from purification sequence la. These inhibitor studies involved: investigating the effect of divalent cations, products of the enzyme reaction and various inhibitors on enzyme activity and the determination of the inhibitor constant (KJ of SAH. These studies involved the incubation of the enzyme solution in standard incubation mixes, in the presence of each of the additives tested, followed by the subsequent assay of these incubation mixes for enzyme activity. Standard incubation mixes used contained saturating substrate levels (SAM was present at 60 pM levels, as a mixture of hot and cold SAM, with a hot SAM ratio of 4.65 % and 1 -hydroxycanthin-6-one was present at 230 pM levels). The standard incubation mix also contained optimum protein levels of 30 pg in 100 pi of the DEAE cellulose enzyme solution. These incubation mixes were incubated under optimum conditions for enzyme activity i.e. a temperature of 30 °C, a pH of 7.5 and an incubation time of 45 minutes. Enzyme assays were carried out in triplicate as described in Sub-Section 2.3.3.3 . A 100 % control to which no inhibitor had been added was also assayed for enzyme activity. Enzyme activity or inhibition, in the other incubation mixes were then expressed as a percentage of the 100 % control.

3.4.3.1 The Effect of Divalent Cations on Enzyme Activity Divalent cations are not always known to inhibit enzyme activity. Sometimes they actually enhance activity and sometimes (though rarely with methyltransferase enzymes), they may be required for activity. The effect of some divalent cations on the 1 -HMT enzyme were investigated using the DEAE cellulose enzyme from purification sequence 1 a. The divalent cations tested include magnesium, manganese and copper as their sulphate or chloride salts. Calculated volumes of stock solutions of each of these salts corresponding to final concentrations of 1 and 10 mM in 100 pi of the enzyme solution, were individually pre-incubated with the enzyme solution at 30 °C for five minutes. Saturating levels of the enzyme substrates, SAM and 1 -hydroxycanthin 6-one were then added to obtain standard incubation mixes, containing each of the divalent cations at each of the required concentrations. These incubation mixes were then incubated under the

208 FIGURE 3.4-15 The Effect of Divalent Cations on 1-HMT Activity

o 150 - IZZZI 1 mM EEa 10 mM o Ü 120 - o

— 90 H >* 4—»

S 60 H o < Q) E 30 - >. N C LU 21

M9 SO4 MQCI2 MnCl 2 C 11CI2

Divalent cations at, 1 and 10 mM levels

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

209 previously described optimum conditions for enzyme activity and enzyme assays were then carried out as described in Sub-Section 2 3 3 3 . The results were presented as a bar chart shown in Figure 3.4-15 in which enzyme activity (expressed as a percentage of the 100 % control) was plotted against the concentration of divalent cations. The results presented in Figure 3.4-15, show that pre-incubation of the enzyme with divalent cations present as their chloride or sulphate salts i.e. MgSO^, MgCl2 and MnClj at 1 and 10 mM levels, resulted in a slight inhibition of the enzyme activity by these cations. MgSO^ gave 14 % and 7 % inhibition at 1 and 10 mM levels respectively, MgCl2 gave 19 % and 20 % inhibition and MnCl2 gave 20 % and 26 % inhibition at 1 and 10 mM levels respectively. These levels of inhibition were not considered to be significant since the concentrations used were relatively high and would not normally pertain to basic extraction procedures. There was however significant inhibition on pre-incubation of the enzyme with CUCI2, which gave 42 % and 80 % inhibition at 1 and 10 mM concentrations respectively. On the basis of these results it can be concluded that the methyltransferase enzyme does not require Mg^^ or Mn^^ for activity and Cu^^ is inhibitory to its activity.

3.4.3 2 The Effect of Enzyme Inhibitors on Methyltransferase Activity The inhibitory effect of some well known enzyme inhibitors on 1- hydroxycanthin-6-one methyltransferase activity were studied using the DEAE cellulose eluate from purification sequence la. The inhibitors tested include potassium cyanide (KCN), iodoacetamide (lA), n-ethylmaleimide (NEM) and para-chloromercuric benzoate (p-CMB) at 0.1, 1 and 10 mM concentrations. Calculated volumes from stock solutions of inhibitors corresponding to final concentrations of 0.1,1 and 10 mM in 100 pi of the enzyme solution (containing about 30 pg of protein) were pre- incubated with the enzyme solution at 30 °C for five minutes. This was followed by the addition of saturating levels of the enzyme substrates; SAM and 1 -hydroxycanthin- 6-one, to obtain standard incubation mixes which were incubated under the previously described optimum conditions for enzyme activity. These incubation mixes contained

210 FIGURE 3 .4 -1 6 The Effect of Inhibitors on 1-HMT Activity

90 - □ 0.1 mM IZZl 1 mM o E E 10 mM c 72 - o o

O 54 -

c o 36 - jQ x : c

lA NEMKCN p—CMB

Inhibitors at 0.1, 1 and 10 mM Levels

Inhibitors used include: potassium cyanide (KCN), iodoacetamide (lA), N-ethyl maleimide (NEM) and p-chloromercuric benzoate (p-CMB) The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

211 each of the inhibitors at each of the required concentrations. They were then assayed for enzyme activity as described in Sub-Section 2.3.3.3 . The results were presented as a bar chart shown in Figure 3.4-16, in which inhibition of enzyme activity (expressed as a percentage of the 1(X) % control) was plotted against inhibitor concentrations. Of the enzyme inhibitors tested, only IA and p-CMB, showed inhibition of over 50 %. lA gave 70 % inhibition of enzyme activity at 10 mM levels and p-CMB gave 75 % inhibition of enzyme activity at 10 mM levels. Since these two inhibitors act on sulphydryl groups, this signifies a strong sulphydryl group involvement in enzyme activity.

3.4.3 3 The Effect of Reaction Products on Enzyme Activity The products of the méthylation reaction of l-hydroxycanthin-6-one by the 1- HMT enzyme in the presence of SAM as the methyl group donor, are 1- methoxycanthin-6-one and S-adenosylhomocysteine (SAH), as shown in Figure 3.4-17. The inhibitory effects of these products on the 1-HMT enzyme were investigated, using the DEAE cellulose eluate from purification sequence la. Standard incubation mixes containing each of these products investigated at each of the required concentrations were then prepared as follows: Volumes from stock solutions of SAH and 1-methoxycanthin-6-one, equivalent to final concentrations of 0.1, 1 and 10 mM in 100 pi of the enzyme solution (corresponding to optimum protein levels of about 30 pg), and saturating levels of the enzyme substrates were concurrently added to the enzyme solution. These standard incubation mixes were then incubated under the previously described conditions for optimum enzyme activity and then assayed for enzyme activity as described in Sub-Section 2.3.3.3. The results were presented as a bar chart (Figure 3.4-18) in which inhibition of enzyme activity (expressed as a percentage of the 100 % control) was plotted against product concentration. The results presented in Figure 3.4-18 show that SAH produced significant inhibition of over 50 % at 1 and 10 mM levels. It gave 89 % inhibition at 1 mM levels and 95 % inhibition at 10 mM levels, l-methoxycanthin-6-

212 FIGURE 3.4-17 The Méthylation of l-Hydroxycanthin-6-one to l-Methoxycanthin-6-one, with SAM as Methyl Group Donor

O II

O C CH CH 2 CH 2 S+ - Adenosyl I I NH2 CH3

SAM to ^ l-Hydroxy-C-6-one ------^ ► l-Methoxy-C-6-one O II O C CH CH; CH; S - Adenosyl I I NH; H

SAH FIGURE 3.4-18 The Effect of the Reaction Products (SAH and 1- Methoxycanthin-6-one) on 1-HMT Activity

100 - □ 0.1 mM EZ] 1 mM 10 mM ^ 80 - o Ü

o 60 - i s c 40 - o

Ic 20 -

SAH 1 —Methoxy—C—Gone

Products at 0.1, 1 and 10 mM levels

The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

214 one however produced a less dramatic inhibitory effect demonstrating significant inhibition, of more than 50 % only at 10 mM levels, where it produced 54 % inhibition.

3.4.3.4 Determination of the Inhibition Constant (Kj) for SAH The inhibition constant (KJ is the inhibition dissociation constant of the enzyme - inhibitor complex (El) and it is a measure of the affinity of the enzyme (E) for the inhibitor (I) in the same sense as the Michaelis-Menten constant is a measure of affinity of the enzyme (E) for the substrate (S). The K, could be determined by using the Dixon plot or the more accurate Lineweaver-Burk plot for either competitive or non-competitive inhibition as the case may be. The inhibitory effect of SAH and l-methoxycanthin-6-one on enzyme activity were studied in Sub-Section S.4.3.3 and based on the results obtained, an attempt was made, to determine the inhibition constants (KJ for both of these products. The DEAE cellulose eluate from purification sequence la was used for these determinations. SAH is expected to inhibit SAM competitively, as a result of their structural similarities (which can be seen in Figure 3.4-17, Sub-Section 3.4.3) and 1- methoxycanthin-6-one is expected to inhibit SAM non-competitively. The Kj of SAH was successfully determined, using both the Dixon and Lineweaver-Burk plots for competitive inhibition. The Kj of 1 -methoxycanthin-6-one could not however be determined due to the poor aqueous solubility of l-methoxycanthin-6-one at levels where it had significant inhibitory activity. This resulted in erratic data which could not be logically interpreted.

3.4.3.4.1 Determination of the Inhibition Constant (KJ for SAH Using the Dixon Plot The Dixon plot is a simple graphical method which requires the determination of the enzyme activity of incubation mixes, at 2 or 3 different substrate concentrations; using a series of inhibitor concentrations at each of the substrate concentrations

215 FIGURE 3.4-19 An Investigation of the Competitive Inhibition of SAM by SAH. The Dixon Plot was used to Determine the K; for SAH

O O CVJ ■ 30 jliM SAM A 60 jjM SAM

o . t : CM > *-> = 4.5 |jM o < o GO Q E >. N C UJ

0.2-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

[SAH mMl

Experiments involved the determination of the inhibitory effect of SAH at 4 concentrations (0, 0.1, 0.5 and 1 mM) on 1-HMT activity at 2 SAM concentrations (30 and 60 pM) The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

216 investigated. This is followed by making a plot of the reciprocal of enzyme activity in (pkat ’) versus the concentration of inhibitor ([I]), at each of the substrate levels investigated. The plot gives a series of straight lines (one for each substrate concentration used). These lines intersect, on or above the abscissa at the value of: [I] = -Kj. The non-competitive intersection point is on the abscissa, while the competitive intersection point is above the abscissa (Michal, 1983), as shown in Figure 3.4-19.

The Kj of SAH was obtained by performing experiments to determine the inhibitory effect of SAH, on enzyme activity at two concentrations of SAM (30 and 60 |iM). These determinations were done, using four different concentrations of the SAH (0, 0.1, 0.5 and 1 mM), at each of the two SAM concentrations chosen. Stock solutions of the inhibitor (SAH) and the substrate (SAM), were prepared as previously described in Sub-Sections 2.3.3.2 and 2.3.3.1 respectively. Volumes of the stock solutions of SAH and SAM necessary to produce each of the required concentrations, in the final incubation mix, were concurrently added to standard incubation mixes containing saturating levels of l-hydroxycanthin-6-one. Incubations were carried out under optimum conditions for enzyme activity and this was followed by assays for enzyme activity as described in Sub-Section 2.3.3.3 . All determinations were done in triplicate. The results are shown in Figure 3.4-19, in which the reciprocal of the enzyme activity in pkat ’ was plotted against concentrations of SAH for each of the chosen SAM concentrations. With the aid of this plot, it was possible to show that SAH competitively inhibited SAM and also to determine the Kj of SAH as 4.5 pM.

3.4.3.4.2 Determination of the Inhibitor Constant (Kj) for SAH Using the Lineweaver-Burk Plot Kj can be readily determined from the Lineweaver-Burk double reciprocal plot for competitive inhibition. This involves studying the enzyme reaction in the presence of a series of substrate concentrations and a fixed inhibitor concentration. This is then repeated at 2 or more inhibitor concentrations and plots of the reciprocal of enzyme

217 activity (1/V), versus the reciprocal of SAM concentrations 1/[S], are made for each of the chosen inhibitor concentrations. These plots result in a straight line obtained for each inhibitor concentration used. These straight lines have different slopes. In the case of competitive inhibition, they intersect on the ordinate axis, as shown in Figure 3.4-20, while in the case of non-competitive inhibition, they intersect on the negative abscissa (Michal, 1978; 1983). With competitive inhibition, the intercept on the abscissa, 1 / [S] is determined as shown in Equation 3.4-105.

Intercept on

1 / [S] = ______.... Equation 3.4-105

Km (1 + [I] / Kj)

With noncompetitive inhibition, the intercept on the ordinate, 1 / V is determined as shown in Equation 3.4-106.

[intercept on , ^ p] /

1 / V =______.... Equation 3.4-106

Experiments were carried out to determine the inhibitory effect of SAH on enzyme activity at three different concentrations of SAH; (0, 0.3 and 20 mM). These determinations were done using 4 different concentrations of SAM, (31, 46, 92, and 140 pM), at each of these three SAH concentrations chosen. Stock solutions of the inhibitor (SAH) and the substrate (SAM) were prepared as previously described in Sub-Sections 2.3.3.2 and 2.3.3.1 respectively. Volumes of the inhibitor (SAH) corresponding to the first of the chosen concentrations (0 mM) and volumes of the substrate (SAM) corresponding to each of all of the four chosen concentrations were added concomitantly to individual standard incubation mixes, each containing

218 FIGURE 3,4-20 An investigation of the Competitive Inhibition of SAM by SAH. The Lineweaver-Burk Plot was used to Determine the K: for SAH

5 0 0 - • 0 mM SAH ■ 0.3 mM SAH T 20 mM SAH I*-• (O 4 0 0 - . 1 Q.

>• Kj = 5 )jM .t: 3 00 - ’> Vm.x = 0,13 .1 O < 200 - 0> E >• N C 100 - UJ

0.000 0.007 0.014 0.021 0.028 0.035 [SAM pM] -1

Experiments involved the determination of the inhibitory effect of SAH at 3 concentrations (0, 0.3 and 20 mM) on 1-HMT activity at 4 SAM concentrations (31. 46. 92 and 140 pM). The assay was done under standard conditions using active fractions from the DEAE-Cellulose column in purification sequence 1

219 saturating levels of 1-hydroxycanthin-6-one. Incubations were carried out under optimum conditions for enzyme activity and this was followed by assays for enzyme activity. This procedure was then repeated at each of the other chosen SAH concentrations, 0.3 and 20 mM. All the determinations done in these set of experiments were in triplicate. A graph of the reciprocal of enzyme activity in pKaf* versus the reciprocal of SAM concentration was plotted for each of the chosen SAH concentrations as shown in Figure 3.4-20. With the aid of this plot, it was possible to show that SAH competitively inhibited SAM and also to determine the of SAH as 5 pM.

Kj determination was done using 2 different methods for the purpose of comparison. Kj values obtained from both methods were virtually equal.

3.4.4 Determination of the Molecular Weight of the 1-HMT Enzyme The molecular weight determination of the 1-HMT methyltransferase enzyme, was done by employing the purest form of the enzyme available i.e. the Superose column eluate from purification sequence la. Figure 3.3-5 in Sub-Section 3.3-1, shows the elution profile of proteins from the superose column, with the blocked peak representing the active fractions, bulked for use in molecular weight determinations. The fact that enzyme activity coincided with a single protein peak (the blocked peak in Figure 3.3-5), suggested that the enzyme had been purified to a single protein. The molecular weight of the enzyme was determined, using two different methods for purposes of comparison. The first method involved gel filtration on a Pharmacia Superose 12 HR 10/30 column and the second method involved, vertical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was also used to confirm the purity of the enzyme.

220 3.4.4.1 Molecular Weight Determination by Gel Filtration on a Pharmacia Superose 12 HR 10/30 Column Gel filtration of the enzyme on a Pharmacia Superose 12 HR 10/30 column served dual purposes of: further purification of the Mono-Q eluate from purification sequence la and molecular weight determination of the superose column eluate. A mixture of molecular weight markers was injected unto the column for purposes of calibration as described in Sub-Section 2.3.6.1.1. This was followed by the injection of 200 pi of the Mono-Q column eluate from purification sequence 1 a. The column was run under similar conditions in both cases and the experimental procedure involved is described in detail in Sub-Section 2.3.5.S . Figure 3.3.5, shows a typical elution profile of the purified 1-HMT enzyme, from the Superose column, where the blocked peak represents the active fractions. The elution volume of this peak was determined as 27.4 ml. Figure 3.4-21, shows the elution profile of standard molecular weight markers from the Superose column where peaks a, b, c and d represent aldolase, egg albumin, chymotrypsinogen A and cytochrome C of molecular weights 158,000; 45,000; 25,000 and 12,000 Dalton respectively. The elution volume of each of the markers was determined and a calibration curve of the molecular weight of the markers versus the elution volume in ml was plotted. The molecular weight of the purified 1-HMT enzyme was subsequently determined as 63,000 ± 2000 Dalton, by interpolation from the standard curve.

221 FIGURE 3.4-21 The Elution Volume of Protein Standards from the Pharmacia Superose 12 HR 10/30 Column, Using Running Buffer B(l).

M olecular Weight (Dalton)M. Wt. Marker Molecular Weight (Dalton)M.

A ld o la s e 158,(XX)

Egg Albumin 45,000

Chymotrypsin 25,000

e Cytochrome C 12,500 c o to oo to CN to 0 C3c 1 CO < > :D

10 20 30 40

Volume (ml) 3AA.2 Molecular Weight Determination by SDS-PAGE Vertical SDS-PAGE was used to confirm the molecular weight of the purified protein solution, which was previously obtained by gel filtration on the Pharmacia Superose 12 HR 10/30 column. The experimental procedure involved, is explained in detail in Sub-Section 2.3.6.1 . The migratory profile of the purified enzyme as well as the mixture of protein standards is shown in Figure 3.4-22. This Figure also shows that the purified enzyme on electrophoretic migration, produced a single protein band thus confirming the fact that the enzyme had been purified to a single protein. The molecular weight markers represented as a, b, c, d, e and f in Figure 3.4-22, are carbonic anhydrase, egg albumin, bovine serum albumin, phosphorylase B, p-galactosidase and myosin of molecular weights 29,000; 45,000; 66,000; 97,000; 116,000 and 205,000 Dalton respectively. A calibration curve of the molecular weights of each of the markers versus the distance migrated by each marker (in mm) from the cathodic end of the gel was subsequently plotted. The molecular weight of the single protein band obtained for the purified 1 -HMT enzyme, was subsequently determined by interpolation from the calibration curve. The molecular weight corresponding to this protein band is 60,000 ± 200 Dalton. The molecular weight obtained by SDS-PAGE, 60,000 Dalton is similar to the molecular weight of the enzyme determined by gel filtration (63,000 Dalton).

223 Figure 3.4-22 The Migratory Profile of the Purified 1-HMT Enzyme of A. altissima during SDS-PAGE

S-26 S-27 S-28 STD

Legend

M. Wt Marker Molecular Weight (D a lto n )

a Carbonic Anhydrase 2 9 ,0 0 0

b Egg Albumin 4 5 ,0 0 0

c Bovine Serum Albumin 6 6 ,0 0 0

d Phosphorylase B 9 7 ,0 0 0

e p-Galactosidase 1 1 6 ,0 0 0

f M y o sin e 2 0 5 ,0 0 0

STD = Mixture of M.Wt Markers S-26, S-27 and S-28 = Active fractions from Superosc 12HR Column

A 15% SDS-Folyacrylamide gel of 1.5 mm thickness was used under the following conditions; constant current 120 rnA, temperature 14.5 °C, and running buffer pH 8.3, on an LKB-2197 Electrofocusing unit. The gel was stained with coumassie blue.

224 3.4.5 Stability Studies Experiments were carried out to study the stability of the 1-HMT enzyme preparation and also to determine suitable conditions for the storage of the enzyme in order to ensure stability over a reasonable period of time. Experiments were carried out on the Mono-Q enzyme eluate from purification sequence la (Sub-Section 3.3-1). The effect of certain additives and storage conditions on enzyme activity were determined in order to investigate their ability to prevent loss of enzyme activity over a 30 day period. These include: storage of the enzyme at 4 °C, -20 °C and -80 °C (under liquid nitrogen) without any additives; storage of the enzyme at 4 °C, with 1 mM levels of cold SAM, 1 mg . ml ' BSA protein and 2 mM leupeptin and finally storage of the enzyme in 30 % glycerol at -20 °C. Quantities of each of these additives required to give the stated concentrations in a known volume of the enzyme preparation were individually delivered into labelled vials and thoroughly mixed with the enzyme solution prior to storage under the required conditions. The results were presented in graphical form in which the enzyme activity was plotted against the storage time. These results were presented as Figures 3.4-23 (a), (b) and (c). Results presented in Figure 3.4-23 (a), showed that storage of the enzyme at 4 °C, resulted in a loss of more than 50 % of enzyme activity by day 6, after which an even more rapid loss of enzyme activity occurred. Storage of the enzyme preparation at -20 °C and -80 °C (in liquid Ng), resulted in a rapid loss of enzyme activity, as most of the enzyme activity had disappeared by day 3, in both cases. Results presented in Figure 3.4-23 (b), showed that storage of the enzyme preparation in the presence of SAM at 4 °C, resulted in a loss of most of the enzyme activity by day 3. Storage of the enzyme preparation in BSA protein at 4 °C, appeared to slow down loss of enzyme activity, as the enzyme maintained about 50 % of its original activity up to day 6, although there was a fairly rapid loss of activity after that. Results presented in Figure 3.4-23 (c), showed that storage of the enzyme preparation in leupeptin at 4 °C was also quite useful in delaying rapid loss of enzyme

225 FIGURE 3 .4 -2 3 Stability Studies of 1-HMT

(a) a 4*C 20000 - # -20*C V -80*C E o. X» 16000

12000 - u < « 8000 - E >. N UJC 4000 - V V 0 20000 (b) O SAM at 4*C ★ BSA at 4'C E ■oo. 16000 -

12000 u < f> 8000 E

4000 -

15000 - o Leupeptin at 4*C A Glycerol at -20*C E ■oQ. 12000

9000 - U < « 6000 - >>E N C UJ 3000 -

— I— 12 18 24

Time (days)

The enzyme was stored under the following conditions: (a) at 4'C, -20*0 and -80*0 without any additives; (b) at 4 *0 with ImM SAM and 1mg.mF^ BSA protein and {cl at 4*0 with 2mM leupeptin and at - 2 0 * 0 with 30% glycerol.

The assay was done under routine conditions using active fractions from the Mono-Q column in purification sequence 1

226 activity. Storage of the enzyme under this condition, resulted in the enzyme maintaining about 50 % of its activity until day 6, after which there was a rapid loss of activity. Storage of the enzyme in 30 % glycerol at -20 °C, however proved to be quite useful in maintaining enzyme activity over a prolonged period of time as can be observed in this Figure. When the enzyme was stored in 30 % glycerol at -20 °C, enzyme activity remained quite constant without any appreciable loss until day 24, after which about 50 % of enzyme activity was lost between days 24 and 30. Storage of the enzyme in 30 % glycerol at -20 °C appeared to be the most effective method for storage of the enzyme and was therefore used routinely whenever necessary, for enzyme storage over long periods of time i.e. for up to 24 days. For shorter storage periods of 3 days or less, the enzyme was stored at 4 °C without any additives. For storage periods of up to 6 days, the enzyme was stored at 4 °C with 2 mM leupeptin.

3.4.6 Méthylation of Hydroxylated Canthin-6-one Substrates by the Canthin-6- one Methyltransferase Enzymes from Various A. altissima Cell Lines The ability of A. altissima cell lines to methylate hydroxylated canthin-6-one substrates was investigated. Apart from cell lines 1 and 2, two additional cell lines, 3 and 4 were also utilised for these experiments. The substrates investigated include the following canthin-6-one substrates: 1-hydroxycanthin-6-one and lO-hydroxycanthin-6-one. Enzyme preparations used for the assays are the DEAE cellulose eluates obtained (as described in Sub-Section 2.3.5), from cell lines 1, 2, 3 and 4 of A. altissima suspension cultures. These cultures were harvested at day 18 in the growth cycle of the cells. Enzyme assays were carried out with each of the hydroxylated canthin-6-one substrates, in triplicate and the results presented in Table 3.4-10, represent the average of triplicate values.

227 Of all the cell lines studied, only cell line 1 methylated both hydroxylated canthin-6-ones. Cell lines 2, 3 and 4 methylated only 1-hydroxycanthin-6-one, with varying activities.

TABLE 3.4-10 Méthylation of Canthin-6 -one Substrates

by Canthin-6 -one Methyltransferase Enzymes from Various A. altissima Cell Lines

Cell line Enzyme Activity Obtained with Hydroxylated Substrates (dpm) * 1-hydroxy- 10-hydroxy- C-6-one C-6-one 1 12,195.45 6,350.36 2 13,034.50 0 3 1,162.25 0 4 29,296.89 0

Legend: * Average of triplicate values

Assay was done under routine conditions, using the DEAE cellulose eluate from purification sequence la. Cells were harvested at day 18 of the growth cycle.

228 3.5 The CMT Enzyme of A. altissima Cell Suspension Cultures

3.5.1 Partial Purification of the CMT Enzyme The partially purified CMT enzyme was obtained from suspension cultures of A. altissima (Cell line 2) which were harvested at day 16 of the growth cycle. The procedure involved an initial enzyme extraction and protein precipitation with (NH4)2S04, as described in Sub-Sections 2.3.2 and 2.3.4 respectively. This was followed by further purification on a DEAE cellulose column. The fractions obtained from this column were assayed for coumarin methyltransferase activity (using aesculetin and fraxetin as substrates), by the method given in Sub-Section 2 3 3 3 . CMT activity corresponded with the first protein peak eluted from the column along with the void volume, indicating that this enzyme did not bind to the column. The 1 -HMT enzyme on the other hand bound to the column and required a KCl gradient for removal (see Figure 3.3-2, Sub-Section 3.3.1). The DEAE cellulose column therefore has the ability to separate the CMT enzyme from the 1-HMT enzyme. The most active fractions exhibiting coumarin methylating ability were bulked for carrying out further experiments on this enzyme. From results presently available it is unclear whether A. altissima cell suspension cultures contained only one CMT enzyme which methylated both aesculetin and fraxetin or whether the cultures contained two different methyltransferase enzymes, for the méthylation of these two coumarins. In order to verify this, further purification of the enzyme is necessary.

3.5.2 Méthylation of Hydroxylated Coumarin Substrates by the CMT Enzyme from A. altissima Cell Suspension Cultures Enzyme assays were carried out on the partially purified CMT enzyme from A. altissima cell suspension cultures, to investigate the ability of these cultures to methylate coumarins. The coumarin substrates investigated were selected to reflect the coumarin methylating patterns of 2 different coumarin méthylation sequences, which were reported to occur in these cultures (Hay, 1987 and Roberts, 1991). These

229 sequences represented schematically in Sub-Section 3.1.6.2, are shown with full structural details in Figure 3.5-1. The substrates investigated include aesculetin, scopoletin and iso-scopoletin from sequence 1 of coumarin méthylation as well as fraxetin, iso-fraxidin and fraxidin from sequence 2. Enzyme assays were carried out by employing the procedure previously described in Sub-Section 2.3.3.3 and using each of these substrates. Experiments were done in triplicate and the results presented are an average of triplicate values. The results obtained from the méthylation of substrates of sequence 1 of coumarin méthylation, (by the CMT enzyme) are presented in Table 3.5-10. These results indicate that with regards to sequence 1, the enzyme methylates aesculetin (with an enzyme activity of 10,652.55 dpm) to form scopoletin. Scopoletin formation was verified by TLC techniques (Sub-Section 2.2.2.1 and Table 2-13, Sub-Section 2.4.1), and product identification experiments (Sub-Section 3.1.6.2). The results presented in Table 3.5-10 also showed that further méthylation of scopoletin by the CMT enzyme did not occur, but isoscopoletin was further methylated by the enzyme to presumably form scoparone. Isoscopoletin méthylation was however at much lower levels (enzyme activity of 733.27 dpm), in comparison to aesculetin méthylation. These results indicate that méthylation of hydroxyl groups at position C-6 of coumarins occur in A. altissima cell suspension cultures, but not méthylations of C-7 hydroxyl groups. This results suggest that it is unlikely that the dimethylated product of this méthylation sequence, scoparone occurs in A. altissima cell cultures, since isoscopoletin has not been identified in these cultures. The results obtained from the méthylation of substrates of sequence 2 of coumarin méthylation (Figure 3.5-1), by the CMT enzyme, are presented in Table 3.5- 11. In sequence 2, fraxetin was methylated with an enzyme activity of 4443.08 dpm to form isofraxidin . Evidence of isofraxidin formation is given by the TLC and HPLC techniques employed (Sub-Sections 2.2.2.1 and 2.22.2 and Tables 2-13 and 2- 15, Sub-Section 2.4-1) and also by experiments carried out for product identification (Sub-Section 3.1.6.2). Results presented in Table 3.5-11 also showed that:

230 FIGURE 3.5-1 The Méthylation of Coumarin Substrates

Sequence 1

C H 3 O

S c o p o l e t i n C H 3 O ( 7 -hydroxy-6-m ethoxy HO' c o u m a r i n ) 8 1 C H 3 O A e s c u l e t i n S c o p a r o n e { 6 , 7 - d i h y d r o x y HO ( 6 , 7 - d i m e t h o x y c o u m a r i n c o u m a r i n ) CH3O ' ' "-'O ^ o

Isoscopoletin ( 6 - h y d r o x y - 7 - m e t h o x y c o u m a r i n )

Sequence 2

C H 3 O

O C H 3 C H 3 O Isofraxidin C H o O (6,8-dim ethoxy- 7 - hydroxy coum arin) CHiO

F r a x e t i n O C H 3 ( 7 ,8-dihydroxy ■ 6 - m e t h o x y C H 3 O 6 , 7 ,8-trim ethoxy c o u m a r i n c o u m a r i n

C H 3 O

F r a x i d i n 6 , 7 -dim ethoxy-8- hydroxy coum arin

231 TABLE 3.5-10 Méthylation of Coumarin Substrates from Sequence 1, by the Partially Purified CMT Enzyme from A. altissima (Cell line 2)

Enzyme Substrate Méthylation Product Enzyme Activity (dpm)* Aesculetin Scopoletin 10,652.55

Scopoletin - - Isoscopoletin scoparone 733.27

Legend: * Average of triplicate values

Assay was done under routine conditions using the DEAE cellulose eluate from purification sequence la.

TABLE 3.5-11 Méthylation of Coumarin Substrates from Sequence 2, by the Partially Purified CMT Enzyme from A. altissima (Cell line 2)

Enzyme Substrate Méthylation Product Enzyme Activity (dpm)* Fraxetin Isofraxidin 4443.08

Isofraxidin - - Fraxidin 6,7,8-trimethoxy 289.43 coumarin

Legend: * Average of triplicate values

Assay was done under routine conditions using the DEAE cellulose eluate from purification sequence la.

232 whilst further méthylation of isofraxidin by the CMT enzyme was not observed, fraxidin was further methylated by the enzyme to presumably form 6,7,8- trimethoxycoumarin. This méthylation was however at much lower levels (enzyme activity of 289.43 dpm), in comparison to fraxetin méthylation. These results indicate that apart from the méthylation of hydroxyl groups at the C-6 position, méthylation of hydroxyl groups at the C-8 position are also favoured by A. altissima cell suspension cultures. These results also further support the idea that méthylation of C- 7 hydroxyl groups, do not occur in A. altissima cell suspension cultures. These results also suggest that it is unlikely that the dimethylated product of this méthylation sequence, 6,7,8-trimethoxycoumarin is present in cell cultures of A. altissima, since fraxidin has not been identified in these cultures.

3.5.3 Méthylation of Coumarin Substrates by Metbyltransferases from Various A. altissima Cell Lines The ability of various cell lines of A. altissima cultures to methylate coumarin substrates were investigated. The coumarin substrates tested include aesculetin and fraxetin, which were selected because they are the starting materials for the 2 sequences for coumarin méthylation (shown in Figure 3.5-1, Sub-Section 3.5.2) reported to occur in A. altissima cell cultures (Hay, 1987, Roberts, 1991). The enzyme preparations used for the assays were obtained from the

DEAE cellulose eluates, obtained from four different cell lines of A. altissima cell suspension cultures. Apart from cell lines 1 and 2, two other cell lines, (3 and 4) were also used. These cell lines (cell lines 1, 2, 3 and 4), were harvested at day 16 in the growth cycle and enzyme preparations obtained from each of them, assayed for activity using each of the hydroxylated coumarins. Experiments were carried out in triplicate and the results presented, represent the average of triplicate values.

All the cell lines investigated exhibited varying abilities to methylate the coumarins aesculetin and fraxetin as shown in Table 3.5-12. Cell line 1 had a significant ability to methylate fraxetin (with an enzyme

233 activity of 18,241.26 dpm) and l-hydroxycanthin-6-one (12,195.45 dpm as shown in Table 3.4-10, Sub-Section 3.4-6), but not aesculetin (1120 dpm). Cell line 2 had a remarkable ability to methylate aesculetin (10,161.59 dpm and 1-hydroxycanthin-6-one (13,034.5 dpm. Table 3.4-10, Sub-Section 3.4.6) but not fraxetin which was methylated at a considerably lower level of 3884 dpm. Cell line 3 on the other hand methylated fraxetin remarkably (14,378 dpm), but methylated aesculetin rather poorly (2976 dpm). l-hydroxycanthin-6-one was also poorly methylated (1162.25 dpm, as shown in Table 3.4-10 of Sub-Section 3.4.6). Cell line 4 had rather poor coumarin methylating ability with enzyme activities of 7233.18 and 2379.24 dpm for aesculetin and fraxetin méthylation respectively. This cell line however had a much more remarkable ability to methylate 1- hydroxycanthin-6-one (29,296.89 dpm. Table 3.4-10, Sub-Section 3.4-6).

TABLE 3.5-12 Méthylation of Coumarin Substrates by CMT Enzymes from Various A. altissima Cell Lines

Cell line Enzyme Activity obtained with hydroxylated substrates (dpm) * Aesculetin Fraxetin 1 1,120.0 18,241.26 2 10,161.59 3,884.0 3 2,976.0 14,378.0 4 7,233.18 2,379.24

Legend: * Average of triplicate values

Assay was done under routine conditions, using the DEAE cellulose eluate from purification sequence la. Cells were harvested at day 16 of the growth cycle.

234 4. DISCUSSION AND CONCLUSIONS

235 4.1 The I-Hydroxycanthin-6 -one Methyltransferase Enzyme (1-HMT) of A. altissima Cell Suspension Cultures A methyltransferase enzyme which catalyses the méthylation of 1- hydroxycanthin-6-one has been isolated from cell suspension cultures of A. altissima. Although this enzyme presently has no known commercial value, it may prove to find application in the general méthylation of compounds or the méthylation of specific groups of compounds. An investigation into this possibility would require a detailed study of the methylating ability of these cultures, as well as further studies involving the isolation and characterisation of the methyltransferase enzyme. These studies subsequently need to be extended to investigations of the specificity of action of the methyltransferase enzyme, studies of the mode of action and possible ways of widening the scope of activity of the enzyme. This project encompasses a preliminary study, in which 1-HMT was isolated and characterised.

4.1.1 The Possible Application of 1-HMT in the General Méthylation of Compounds General méthylation reactions are carried out chemically by the action of several toxic agents, which include the following amongst others: Phenyl sulphonyl magnesium bromide (for the méthylation of carboxylic esters with hydroxyl groups), dimsylsodium (sodium methylsulphinylmethide) in DMSO (glycosides of polysaccharides), (Fieser and Fieser, 1969), dimethylsulphonium methyl ide in THF (indoles), (Fieser and Fieser, 1974), methylfluorosulphonate (amines, amides, nitriles and esters), methylene bromide and magnesium amalgam (carbonyl compounds), dimethylsulphonium methyl ide (acidic NH and OH groups and some aromatic hydrocarbons), (Fieser and Fieser, 1972), sodium hydride and methyl iodide in THF (phenols and alcohols) and trimethyl phosphate (thymine and uracil), (Fieser and Fieser, 1975). The méthylation of compounds by methyltransferase enzymes, offers the distinct advantage of being a less toxic method than the chemical methods mentioned

236 above. However, as in the case of biotransformation reactions, in order for this process to be a commercial reality it must be economically viable and not be in competition with the chemical methods of méthylation. A major drawback to the application of methyltransferase enzymes in general méthylation reactions or in the méthylation of specific groups of compounds is their specificity of action. Most metbyltransferases studied in literature have been reported to be highly specific in action. N-methylating enzymes in particular are usually highly specialised. Most secondary metabolites containing N-groups, which have been reported to act as substrates for N-methyltransferases are usually alkaloids or xanthines (Sub-Section 1.3.2). Several 0-methyltransferase enzymes studied in literature have also been reported to show specificity of action. Examples include: Caffeoyl CoA-3- 0-MT from Daucus carota cell suspension cultures (Kunhl et al., 1989); Caffeic acid- 3-O-MT from Medicago sativa cell suspension cultures (Edwards and Dixon, 1991); 3-Methylquercetin-7-0-MT from Chrysosplenum americanum shoot tips (Khouri et at., 1988 a) and 3'-hydroxy-N-methyl (S)-coclaurine-4'-0-MT from Berberis koetineana cell cultures (Frenzel and Zenk, 1990 b). O-Methyltransferases generally exhibit a wider range of activity than the N-methyltransferases. They are reportedly involved in the metabolism of lignin, phenolic compounds, flavonoid compounds, phenyl propanoids, furanocoumarins and alkaloids (Poulton, 1981). O-Methyltransferases by virtue of their wider range of activity may be potentially useful for carrying out general méthylation reactions or the méthylation of specific groups of compounds on a commercial scale. 1-HMT was thought to be an ideal model for carrying out this investigation since cell cultures of A. altissima (from which 1-HMT was extracted), were relatively easy to maintain and also grew rapidly. However, 1-HMT like most metbyltransferases, demonstrates specificity of action. Although this has not been studied in detail, preliminary studies showed that: When four cell lines of Ailanthus altissima (cell lines 1, 2, 3 and 4), were investigated for their ability to methylate 1- and lO-hydroxycanthin-6-one, only cell line 1 demonstrated the ability to methylate both 1-hydroxycanthin-6-one and lO-hydroxycanthin-6-one. All the other cell lines

237 only methylated l-hydroxycanthin-6-one (Table 3.4-10, Sub-Section 3.4.6). Roberts et al. (1989) and Roberts (1991), also reported that 1-hydroxycanthin- 6-one, lO-hydroxycanthin-6-one and 8-hydroxycanthin-6-one methyltransferase activities were separated in a cell line of A. altissima, which demonstrated the ability to methylate all three hydroxylated canthin-6-ones.

4.1.2 The Methylating Ability of A. altissima Cell Suspension Cultures: The

Méthylation of I-Hydroxycanthin-6 -one The methylating abilities of 2 cell lines of A. altissima (cell lines 1 and 2), were studied in order to have a detailed knowledge of the growth cycle of the cells. The growth, canthin-6-one alkaloid production and 1-HMT enzyme activity exhibited by these cell lines throughout their growth cycles were investigated. From these experiments, certain observations and deductions were made which are summarised in Sub-Section 4.1.2.1. Details of these observations can be found in Sub-Section 3.2.1.

4.1.2.1 A Comparison of the Canthin-6 -one Methylating Capacities of Cell Lines 1 and 2: A Summary of the Observations 1. Growth of Cells Both cell lines 1 and 2 underwent a 5 fold increase in fresh weight over the growth cycle, as shown in Figures 3.2-1(c) and 3.2-2(c), Sub-Section 3.2.1, respectively.

2. Alkaloid Production (i) Alkaloid production was more prominent in cell line 1 (Figure 3.2-1 (a), Sub- Section 3.2-1) than cell line 2 (Figure 3.2-2(a), Sub-Section 3.2.1). A comparison of the total alkaloid content of cell lines 1 and 2, (as the cells were about to enter their stationary phases), showed that cell line 1 produced total alkaloid levels of about 100 mg . g dry wt of cells ' and cell line 2 produced only 30 mg. g dry wt. of cells '. (ii) 1 -Hydroxycanthin-6-one production was low and at similar levels throughout the

238 growth cycles in both cell lines 1 and 2. (iii) Cell lines 1 and 2 produced similar levels of canthin-6-one. However in cell line 1, there were no obvious peaks in canthin-6-one levels, whilst in cell line 2, a definite peak of canthin-6-one levels, occurred at days 22 to 30 of the growth cycle. (iv) Cell line 1 produced significant levels of l-methoxycanthin-6-one (5 to 70 mg . g dry wt. of cells’), whilst cell line 2 produced very low levels of the methylated canthin-6-one (1 to 6 mg . g. dry wt. of cells ’). (v) In cell line 1, the rapid increase in l-methoxycanthin-6-one production per unit of cell weight., coincided with rapid cell multiplication. Maximum l-methoxycanthin-6- one levels were obtained towards the end of the growth cycle (when the cells had multiplied 5 times in weight; just before they entered into their stationary phase). This phenomenon is not however apparent with the formation of canthin-6-one or 1- hydroxycanthin-6-one. In cell line 2 however, 1-methoxycanthin-6-one production was very low and did not correlate with cell multiplication. Canthin-6-one levels on the other hand peaked towards the end of the growth cycle, as the cells entered into the stationary phase, whilst 1-Hydroxycanthin-6-one levels remained at similar levels throughout the growth cycle.

3. 1-HMT Activity (i) Maximal 1 -HMT activity was at similar levels in both cell lines 1 and 2. In cell line 1, I-HMT activity was prominent throughout the growth cycle. (Figure 3.2-1(b), Sub-Section 3.2.1). In cell line 2, 1-HMT activity was also prominent throughout most of the growth cycle, except for the period between days 24 and 28, where there was an almost complete loss of methyltransferase activity (Figure 3.2-2(b), Sub- Section, 3.2.1). (ii) In cell line 1, where efficient méthylation of 1-hydroxycanthin-6-one occurred, 1- HMT activity was at peak levels at the time of the growth cycle when 1-methoxycanthin-6-one was produced at maximum levels (days 20 to 22). In cell line 2 however, 1-HMT activity was minimal at the time in the growth cycle when production of l-methoxycanthin-6-one was expected to be maximal i.e. as the cells

239 entered the stationary phase (Roberts et al., 1989), (the stationary phase occurred between days 20 to 28). In this cell line however, canthin-6-one levels peaked at this time in the growth cycle.

4.1.2.2 The Divergence in Methylating Abilities of Cell Lines 1 and 2 of A. altissima: l-Hydroxycanthin-6-one Méthylation Cell lines 1 and 2 of A. altissima were grown under similar conditions but possess different abilities to methylate l-hydroxycanthin-6-one. (Cell line 1, Figure 3.2-1, Sub-Section 3.2.1 produced 10 times more 1-methoxycanthin-6-one than cell line 2, Figure 3.2-2, Sub-Section 3.2.1). There was also a disparity in the total alkaloid content of both cell lines; The overall production of alkaloids in cell line 1 was significantly higher (3.5 times) than cell line 2. The efficient production of 1- methoxycanthin-6-one by cell line 1, as opposed to cell line 2, was mainly responsible for the much higher yields of alkaloid in cell line 1 than cell line 2. Several factors may be responsible for the difference in methylating abilities of cell lines 1 and 2. A study of these determinants was outside the scope of this project, however the possible determinants have been discussed. The difference in methylating abilities of cell lines 1 and 2, may be due to one or more of the following factors:

1. 1-HMT Activity 1-HMT activity may play a partial role in the ability of these cell lines to methylate l-hydroxycanthin-6-one. This is suggested by the fact that Cell line 2, which had poor methylating activity, also exhibited poor 1-HMT activity at days 20 to 28 of the growth cycle, i.e. the onset of the stationary phase, when maximal 1- methoxycanthin-6-one production was expected (Roberts et al., 1989). Cell line 1 on the other hand had significant 1 -HMT activity at the corresponding time in its growth cycle, when it produced maximum levels of 1 -methoxycanthin-6-one.

240 2. Feed back inhibition by l-Hydroxycanthin-6-one or other intermediates of the biosynthetic pathway If 1 -hydroxycanthin-6-one does not undergo further méthylation, it may inhibit the hydroxylase enzyme responsible for its production in the canthin-6-one alkaloid biosynthetic pathway. The constant low levels of l-hydroxycanthin-6-one observed throughout the culture growth cycle, suggests that hydroxylase activity may in part regulate the canthin-6-one alkaloid biosynthetic pathway. Feed back inhibition by 1- hydroxycanthin-6-one is however unlikely to play a significant role in the poor methylating ability of cell line 2, since similar levels of l-hydroxycanthin-6-one occur in both cell line 1 (where significant méthylation occurs) and cell line 2. However it is possible that the proficient production of l-methoxycanthin-6-one by cell line 1, may play a role in the prevention of feedback inhibition by 1 -hydroxycanthin-6-one in this cell line. It is also possible that feed back inhibition is in operation at a much earlier stage in the biosynthetic pathway i.e. either (a) the conversion of tryptophan to P- carboline-1-propionic acid, (b) the conversion of p-carboline-1-propionic acid to 4,5- dihydrocanthin-6-one or (c) the conversion of 4,5-dihydrocanthin-6-one to canthin-6- one. Each of these steps would however have to be further investigated in detail in both cell lines, to substantiate this suggestion. It should however be noted that, these intermediates have not been detected in cell lines of A. altissima grown in our laboratory, even when large scale extractions were carried out (Buchar, F., 1993, unpublished results). Of all the documented studies on A. altissima cell cultures, only Crespi-Perellino et al., (1986 a,b) reported the presence of these intermediates, in very low levels.

3. The location of alkaloids The fact that both cell lines 1 and 2, produced similar levels of 1- hydroxycanthin-6-one, but varied in their ability to methylate this alkaloid, raised questions on the possibility of the location of alkaloids playing a role in the

241 methylating ability of these cultures. During the large scale extraction of l-hydroxycanthin-6-one and other canthin- 6-one alkaloids from the cells, unexpected difficulties were encountered (Roberts, M.F., personal communications), which indicated that the alkaloids were not readily extractable. The difficulty in accessibility to l-hydroxycanthin-6-one may be because the alkaloids exist as salts, which are poorly soluble in Chloroform and methanol used for extraction. However it is also possible that this inaccessibility, (which may also make 1-hydroxycanthin-6-one unavailable as a substrate for the 1-HMT enzyme), may be due to the location of the alkaloids. The possible location of alkaloids within cell cultures of A. altissima will subsequently be discussed. (i) Location of Alkaloids within the Vacuole Hay (1987), reported that alkaloids of A. altissima may partly reside within the vacuoles. Vacuolar sequestration of vindoline has been reported to occur in Catharanthus roseus cell suspension cultures. These cultures do not synthesise vindoline, however they contain a highly specific vacuolar uptake system for this alkaloid (Deus-Neumann and Zenk, 1984; De Luca and Cutler, 1987). Vacuolar sequestration following hydroxylation may also occur with canthin-6- one alkaloids of A. altissima cell cultures. This may be partly responsible for (a) the poor accessibility to the hydroxylated alkaloid during extraction and (b) the poor methylating ability observed in cell line 2 of A. altissima cell cultures. Vacuolar sequestration on the other hand may not affect the availability of 1- hydroxycanthin-6-one, since alkaloids present in the vacuoles are expected to be easily released upon plasmolysis of the cells. (ii) Compartmentation of Alkaloids Compartmentation of protoberberine alkaloids has been reported to occur in cell cultures of Berberis species (Hinz and Zenk, 1981; Rueffer et al., 1990). A similar compartmentation of alkaloids may occur in cell cultures of Laburnum alpinum. These cultures possess a relatively active SAM: cytisine N-MT activity and are able to methylate exogenous cytisine in vivo (Wink, 1984). Cytisine and N-

242 methylcytisine were however not found in cell cultures of L. alpinum (Wink et al., 1983). A similar compartmentation of hydroxylated canthin-6-one alkaloids in A. altissima may reduce availability of the alkaloids, thereby limiting the methylating ability of the cultures. (iii) Location of alkaloids within the Cells Shikonin particles produced by cultured cells of Lithospermum erythrorhizon have been reported to be located between the plasma membrane and cell walls, as confirmed by electron microscopy (Tsukada and Tabata, 1984; Shimomura et al., 1991). It is thought that these shikonin particles which cover the cells, may inhibit cell growth and the further production of shikonin derivatives by interfering with the supply of oxygen and nutrients to the cultures (Deno et al., 1987). Location of 1-hydroxycanthin-6-one within the cell walls may also occur in A. altissima cell cultures, thereby limiting its availability. This would however have to be verified.

4. Availability of SAM The availability of SAM in cell line 2 of A. altissima cell culture, is another factor which may play a partial or full role in the poor methylating ability of this cell line. SAM is the primary donor of methyl groups in transmethylation reactions and its availability is apparently dependent on its effective biosynthesis in cells, where it is formed directly from ATP and methionine. The requirement of SAM in primary metabolism, may be in competition with its role in secondary product méthylation. The availability of methionine and ATP could also play regulatory roles in méthylation, due to their requirement in primary metabolism. There is evidence that SAM is the methyl donor of phosphatidylcholine in plants and animals (Bowman and Rohringer, 1970; Livesey, 1984; Davies, 1987 b). Feeding experiments indicated that phosphatidylcholine is the precursor of choline.

243 Choline is a major phospholipid in cellular membranes and it is involved in the synthesis of cellular membranes and the méthylation of y-aminobutyricacid to y- butyrobetaine (Hitz et al., 1981). L-Camitine, which is involved in the transport of acyl groupings across biological membranes is obtained by the oxidation of y- butyrobetaine (Bohinski, 1979 b). ATP is utilised in nucleic acid biosynthesis (Bohinski, 1979 c) and plays a crucial role in energy metabolism (Lipmann, 1941; Davies, 1987 a) Methionine is involved in the synthesis of ethylene from the amino acid 1- aminocyclopropane-1 -carboxylase (ACC) via SAM (Yang and Hoffman, 1984). Ethylene regulates many aspects of plant growth including germination and senescence (Yang and Hoffman, 1984). The possible regulatory role of methionine is substantiated by experiments carried out by Anderson et al., 1987 b, in which L-methionine was fed to cell suspension cultures of A. altissima. These results suggested that L-[methyl '"^C]- methionine fed in the early stages of growth is in competition with higher native levels of methionine (since low incorporation of the ‘"‘C-label was reported), whereas in the latter stages of the growth cycle, this amino acid is in short supply (since high incorporation of the "‘C-label occurred) (Roberts et al., 1989). If a similar situation to this occurred in cell line 2 i.e. (low native pools of methionine) at the stage where maximum méthylation was expected, it could be responsible for the poor méthylation of l-hydroxycanthin-6-one observed towards the end of the growth cycle in this cell line.

5. SAH levels SAH is the product of all transmethylation reactions involving SAM. SAH strongly inhibits most transmethylation reactions by acting competitively with SAM at low concentrations and the K, for SAH may be several fold lower than the for SAM (Poulton and Butt, 1975; Wegenmayer et al., 1974). The low Kj value for SAH (4.5 - 5.3 pM) in comparison to the for SAM (18-24 pM), with 1-HMT, suggests that SAH powerfully inhibited 1-HMT activity. If this inhibitory activity is more

244 pronounced in cell line 2, it may be partly responsible for the poor méthylation observed in cell line 2. Poulton and Butt (1975), suggested that transmethylation reactions may be controlled by the intracellular SAM : SAH concentration ratio. The precise control mechanism involved is unknown, however it is obvious from the powerful inhibition of transmethylation reactions by SAH, that the removal of this product or the strict regulation of its intracellular concentration is necessary for the continuation of transmethylation reactions. (Poulton, 1981). SAH is further metabolised in plants by SAH hydrolase to yield homocysteine and adenosine. This enzyme also catalyses the reversible synthesis of SAH from adenosine and L-homocysteine. Studies on the kinetic properties of the spinach, beet and lupine SAH hydrolase enzymes, showed that equilibrium lay heavily in favour of SAH synthesis. Sustained SAH hydrolysis is therefore governed by disposal of the products, adenosine and L-homocysteine. In addition to this, accumulation of either of the products reportedly inhibited the enzyme (Poulton and Butt, 1976; Guranowski and Pawalkiewicz, 1977; Poulton, 1981). It is therefore thought that the SAH hydrolase may play a crucial role in metabolism, by controlling the rate of SAH turnover and thereby indirectly regulating the availability of the methyl group (Poulton, 1981).

6. Further metabolism of l-methoxycanthin-6-one There is a possibility that 1 -methoxycanthin-6-one is formed in cell line 2, but is rapidly metabolised thereafter. The further metabolism of 1-methoxycanthin-6-one is known to occur in A. altissima (Anderson et a/., 1987 b). However, the fact that a build up of canthin-6-one occurs in cell line 2, reduces the possibility of 1 -methoxycanthin-6-one being further metabolised. Anderson et al., 1986, reported that the feeding of L-[methylene tryptophan into A. altissima cell suspension cultures at day 0 of the growth cycle, resulted in fluctuations in the label incorporated into the alkaloid, during time course

245 studies. These results suggest a complex catabolism (turnover) of alkaloids during the growth cycle (Roberts et al., 1989). Further experiments in which L-[methylene tryptophan was fed into the cell cultures at different times during the growth cycle, were carried out. These results showed that there was low incorporation of L- [methylene -tryptophan, when fed in the lag phase and high incorporation when fed in the linear phase of growth. The incorporation pattern of L-[methylene ‘"‘C] observed in these experiments, suggested that:

(a) There may be changes in metabolic pools of tryptophan, during the growth cycle of A. altissima, with high native pools of tryptophan occurring in the lag phase and low pools of tryptophan in the linear phase of growth (Roberts et al., 1989). (b) Possible variations in activity of key biosynthetic enzymes may occur during the growth cycle. Although tryptophan has been reported to activate tryptophan decarboxylase in microorganisms (Floss et al., 1974), it has been reported to strongly inhibit anthranilate synthase in plants, bacteria and fungi (Poulson and Verpoorte, 1991; Niyogi and Fink, 1992). Anthranilate synthase is involved in an earlier step in the biosynthesis of tryptophan, a precursor in the biosynthesis of indole alkaloids (Figure 1-4, Sub-Section 1.2.4.1.1). The involvement of tryptophan decarboxylase in indole alkaloid biosynthesis has however been questioned by Yeoman (1993), who was unable to demonstrate the incorporation of '"^C-tryptamine into these cultures. Yeoman (1993), also reported that levels of tryptophan decarboxylase detected in these cultures were very low and did not account for the involvement of the enzyme in indole alkaloid biosynthesis.

4.1.3 Purification and Characterisation of 1-HMT The application of 1 -HMT in general méthylation reactions would require its isolation and purification in fairly significant quantities from cell cultures of A. altissima. This would then be followed firstly by an investigation of the scope of specificity of 1-HMT and secondly, by an investigation of the mode of action of 1-

246 HMT and ways in which the active sites can be altered in order to decrease the specificity, thereby increasing the scope. The latter part of the investigation was not carried out in the course of this project. It is however discussed in Section 4.4. Two methyltransferase enzymes named 1-HMT (responsible for the méthylation of 1-hydroxycanthin-6-one) and CMT (responsible for the méthylation of hydroxylated coumarins), were identified in A. altissima cell suspension cultures. These methyl transferase enzymes have been separated and 1-HMT was purified to a single protein and then characterised.

4.1.3.1 The Selection of the Methodology for Purification of 1-HMT Purification of 1-HMT was from cell line 2 of A. altissima cell cultures, which did not demonstrate the ability to methylate the other hydroxylated canthin-6-one tested (lO-hydroxycanthin-6-one). Cell line 2 was selected because the target was to isolate and purify the 1-HMT enzyme and not the lO-hydroxycanthin-6-one methyltransferase enzyme. Cell line 2 is also fast growing and demonstrated high 1- HMT activity. Of the procedures tested for the purification of 1-HMT (described in detail in Section 3.3), purification sequence la was selected for perfunctory use, despite a major disadvantage it exhibited i.e. the carry through of low molecular weight proteins to the final purification step (the Pharmacia Superose 12 HR column). Sequence la was selected on the basis of its ability to purify 1-HMT to a single protein within a reasonable length of time. The major disadvantage, however resulted in a poor loading capacity for the Superose column and consequently a difficulty in obtaining sufficient amounts of the purified protein, to carry out experiments involved with the characterisation of the enzyme. As a result of this, characterisation of 1-HMT was done on the penultimate and pre-penultimate purification steps i.e. the Mono-Q and DEAE cellulose eluates from purification sequence la respectively. Purification sequences 2a and 3a involved the incorporation of the hydroxylapatite column into the purification procedure for 1-HMT. The

247 hydroxylapatite column, as applied in sequence 3a, in combination with the DEAE cellulose column, appeared to have a great potential for getting rid of extraneous proteins. However in order for purification sequence 3a, to attain levels of purification similar to sequence la i.e. (the purification of 1-HMT to a single protein), it would be necessary to incorporate 2 steps, into the procedure (the Mono-Q and the Pharmacia Superose 12 HR column steps). This would make this procedure, a lengthy 6 step one. Purification sequence 3a was therefore rejected on these grounds. Purification sequences 4a and 5 a involved the incorporation of the Sephadex G-50 and Sephacryl S-200 gel filtration steps respectively into the purification procedure for 1-HMT. These purification procedures were introduced in an attempt to rectify the major problem encountered in sequence la (the carry through of low molecular weight proteins to the final purification step). These procedures however proved to be a disappointment. In spite of the use of both gel filtration steps at an earlier stage in the purification, the low molecular weight proteins still posed a problem at the final purification step. This is probably due to the fact that gel filtration columns have sometimes been found to be ineffective for separating proteins, in which the molecular weight of one protein is less than twice the molecular weight of the other.

4.1.3.2 The Comparison of the Characteristics of 1-HMT with Other Metbyltransferases

4.1.3.2.1 The Characteristics of 1-HMT The characteristics of 1-HMT were found to be as follows: 1-HMT was found to be an enzyme of molecular weight 60,000 - 63,000 Dalton. The enzyme was found to be a monomer, as it appeared as a single protein band on SDS- PAGE. 1 -HMT did not require divalent cations, Mg^"^ and Mn^^, for activity and was inhibited by Cu^^. 1-HMT was slightly inhibited by potassium cyanide and strongly inhibited by SH group inhibitors iodoacetamide and p-chloromercuric benzoate. 1-HMT was slightly inhibited by l-methoxycanthin-6-one, but strongly inhibited by

248 SAH, which competitively inhibited SAM, with a Kj of 4 - 5 pM. 1-HMT was found to operate optimally under the following conditions: an incubation time of 45 minutes; a temperature of 35 °C, and a pH of 7.5. 1 -HMT was stable for up to 24 days when stored in 30 % glycerol at -20 °C.

A list of properties of several O- and N-methyltransferases studied in literature were compiled as shown in Tables 1-14 and 1-15 respectively. The characteristics of 1 -HMT were compared with various metbyltransferases listed in these tables. Characteristics of 1-HMT (which will be looked into in this section) were found to bear strong similarities to several of these O and N- methyltransferases.

4.1.3.2.2 Optimum Temperature The optimum temperature of the 1-HMT enzyme was 35 °C (Figures 3.4-5 and 3.4-6, Sub-Section 3.4.1). This compares favourably with the temperature optima of most metbyltransferases studied in literature, which were reported to be at the rather high level of 35 °C to 40 °C, as shown in Tables 1-14 and 1-15. Examples are nicotinic acid N-MT, obtained from soy bean cell suspension cultures (Upmeier et al., 1988) and tetrahydroisoquinoline N-MT, obtained from Berberis koetineana cell cultures (Frenzel and Zenk, 1990 a), which both had a temperature optimum of 35 to 40 °C. S-tetrahydroprotoberberine cis N-MT obtained from Eschscholtzia californica and Corydalis vaginans cell suspension cultures (Rueffer et al., 1990), exhibited a temperature optimum at the higher level of 40 °C.

4.1.3.2.3 Optimum pH The optimum pH of the 1-HMT enzyme fell within a plateau range of 7 to 7.5 (Figures 3.4-9 and 3.4-10, Sub-Section 3.4-1). The optimum pH range of 0.5 of a pH unit is advantageous because slight fluctuations in pH during incubation and enzyme assay would have little effect on the activity of the enzyme. A methyltransferase enzyme which exhibited a similar pH

249 optimum range to 1-HMT is 6a-hydroxymaackian-3-0-MT obtained from Pisum sativum (Preisig et al., 1989), which had a pH optimum within the range of 7.3 - 7.9. In certain cases concerning methyltransferases, deviation of the pH by 1 unit from the optimum value, could result in significant loss of activity, as was reported for isoflavone 5-O-MT from yellow lupin roots (Khouri et at., 1988 b). Methyltransferases studied in literature, were reported to have a pH optima ranging from the lower level of 6.5, reported for caffeic acid 3-0-MT obtained from Beta vulgaris (Foulton and Butt, 1975) to the higher level of 9.7 reported for O- dihydricphenicol meta O-MT obtained from parsley cell suspension cultures (Ebel et al., 1972). Other examples can be found in Tables 1-14 and 1-15. Other methyltransferases with pH optima very closely related to that of A. altissima 1-HMT, include the 3 isoforms of the enzyme tetrahydroisoquinoline N-MT obtained from cell suspension cultures of Berberis koetineana, with optimum pH levels of 7.4, 7.2 and 6.8 as well as an isoflavone 5-O-MT, obtained from Lupinus luteus roots (Khouri et al., 1988 b), with an optimum pH of 7.0.

4.1.3.2.4 Kinetic Properties The Km values of substrates tested on different enzymes cannot be effectively compared, because other factors like the type of substrates and reactions involved), come into play with different enzymes (Bohinski, 1979 a). The Km values of SAM (18 pM, for the Mono-Q eluate. Figure 3.4-12, Sub- Section 3.4.2) and 1 -hydroxycanthin-6-one (22 pM, for the Mono-Q eluate. Figure 3.4- 14, Sub-Section 3.4), had a ratio of 1 : 1, indicating that the two substrates have a similar level of affinity for the enzyme.

250 4.1.3.2.5 Inhibitor Studies

The Effect of Divalent Cations on Enzyme Activity 1-HMT, like the majority of methyltransferases of plant origin, studied in literature did not show any requirement for the divalent cations and Mn^^ (Figure 3.4-15, Sub-Section 3.4.3). 1-HMT was however, significantly inhibited by Cu^^ (Figure 3.4-15, Sub- Section 3.4.3). It is possible that the presence of Cu^^ in the incubation mix, led to the an irreversible binding of Cu^^ to the enzyme’s active sites. If Cu^^ has a greater ability to bind to the enzyme’s active sites than the substrates, it could alter the binding sites, thereby resulting in the pronounced inhibition observed. It is also possible that Cu^^ may act as an allosteric inhibitor; i.e. although Cu^^ does not bind on the primary active site of the enzyme, it may bind on a secondary active site. This could cause a shift in the structure of the protein molecule, which in turn can affect the catalytic activity of the primary active site (Bowman and Rand, 1980). Examples of other methyl transferases reported not to require divalent cations for activity include the following: Putrescine N-MT from tobacco roots (Mizusaki et al., 1971), nicotinic acid N-MT from soy bean cell suspension cultures (Upmeier et ah, 1988), caffeic acid 3-0-MT from Beta vulgaris (Poulton and Butt, 1975), SAM: O-dihydric phenol O-MT from tobacco cell suspension cultures (Tsang and Ibrahim, 1979), isoflavone-4'-0-MT from Cicer arietinum seedlings and cell suspension cultures (Wegenmayer et al., 1974), SAM: 3-methylquercetin-7-O-MT from Chrysosplenum americanum shoot tips (Khouri et al., 1988 a), isoflavone-5-O-MT from Lupinus luteus roots (Khouri et al., 1988 b) and SAM: dopamine-3-O-MT from Lophophora williamsii (Basmadjian and Paul, 1971). The enzyme activity of caffeoyl CoA-3-O-MT, unlike most methyltransferases was however reported to be stimulated slightly by Mg^"^.

The Effect of KCN on Enzyme Activity 1-HMT was slightly inhibited by KCN (Figure 3.4-16, Sub-Section 3.4.3).

251 Another case of CN' inhibition of methyltransferases was reported by Roberts and Waller (1979), who reported that CN' inhibited the méthylation of 7-methylxanthine to theobromine by the enzyme xanthin-N-MT. A pronounced inhibition by CN', was also reported for loganic acid O-MT (Madyastha et al., 1973). The mode of inhibition by CN' is speculative. Madyastha et at. (1973), proposed that CN' may open disulphide bridges by forming thiocyanates.

The Effect of SH Group Inhibitors on 1-HMT 1 -HMT was strongly inhibited by SH group inhibitors iodoacetamide (lA) and p-chloromercuric benzoate (p-CMB) (Figure 3.4-16, Sub-Section 3.4.3). This is a strong indication of the SH group requirement of the enzyme for activity. Further confirmation of the SH group requirement of 1-HMT, was obtained when experiments in which the crude dialysate from Ailanthus altissima cell suspension cultures was tested for its requirement for reducing agent, DTT or sulphydryl group protecting reagent, |3-MCE. Results showed that exclusion of DTT or p-MCE from the incubation mix resulted in a loss of activity (Figure 3.1-1, Sub-Section 3.1.5). This characteristic is likely to arise from readily accessible sulphydryl groups on the enzyme, which are subjected to oxidation by enzymes released upon homogenisation of the plant tissue, in the absence of DTT or p-MCE (Madyastha et al., 1973). Methyltransferases studied in literature are typically sensitive to sulphydryl reagents. Several methyltransferases have been reported in literature to be inhibited by SH group inhibitors. Several methyltransferases studied in literature also showed a requirement for SH protecting groups like the monothiol p-MCE, DTT or reduced glutathione, which are frequently included in extraction buffers during purification procedures and in assay mixtures (Poulton, 1981). Results reported from studies conducted with methyltransferases showed that SH group inhibitors which include p-CMB, NEM and iodoacetamide, demonstrated a significant inhibitory effect on several methyltransferases. The following enzymes were strongly inhibited by p-CMB: Xanthin-N-MT from coffee fruits (Mizusaki et al., 1971); Nicotinic acid N-MT from

252 soybean cell suspension cultures (Upmeier et al., 1988); SAM: O-dihydric phenol O- MT from tobacco cell suspension cultures (Tsang and Ibrahim, 1979) and Isoflavone 4 -O-MT from Cicer arietinum seedlings and cell suspension cultures (Wegenmayer et at., 1974). SAM: 3-Methylquercetin-7-O-MT from Chrysosplenum americanum shoot tips was strongly inhibited by p-CMB and NEM (Khouri et a l, 1988 a) and Isoflavone-5-O-MT from Lupinus luteus roots was inhibited by NEM and lA (Khouri et a l, 1988 b). Loganic acid O-MT from Vinca rosea (Madyastha et al., 1973) was inhibited by all 3 inhibitors. Loganic acid O-MT also showed a requirement for reducing agent DTT; complete inactivation of the enzyme occurred when DTT was omitted from the incubation mix. In a few exceptional cases however SH group inhibitors failed to inhibit methyl transferase activity. For example, p-CMB had no inhibitory effect on the activity of O-dihydricphenicol meta-O-MT from parsley cell suspension cultures (Ebel et al., 1972).

253 4.2 The Coumarin Methyltransferase Enzymes of A. altissima Cell Suspension Cultures The methylated coumarins scopoletin and isofraxidin , have been identified in A. altissima cell cultures. The enzyme responsible for carrying out these méthylation reactions has also been identified and isolated from these cultures. This enzyme was named coumarin methyltransferase (CMT). These observations together with the reports of Anderson et al. (1987 a,b) (that feeding L-[methyl "‘C-methionine] in cell suspension cultures of A. altissima, resulted in incorporation into the methylated coumarin, scopoletin), suggested that the biosynthetic pathway for the production of methylated coumarins existed in A. altissima. Coumarin biosynthesis and its regulation to date has only been poorly investigated. Present knowledge of coumarin biosynthetic pathways is based primarily on precursor studies, employing intact plant or plant parts e.g. the formation of puberulin in Agathosma puherula, was established by in vivo tracer techniques (Brown etal, 1988; Dewick, 1985, 1991). In the course of this project, A. altissima cell cultures were investigated as a tool for studying the biosynthesis of coumarin. Employing plant cell culture techniques in biosynthetic studies would make the isolation of intermediates and enzymes involved in the pathway possible.

4.2.1 A Comparison of the Coumarin Methylating Abilities of 2 cell Lines of A. altissima (Cell Lines 1 and 2): A Summary of the Observations Two cell lines of A. altissima (cell lines 1 and 2) were investigated for their abilities to methylate hydroxylated coumarins. The growth, coumarin methylating abilities and CMT activity of these two cell lines were studied throughout their growth cycles and the results compared. Details of these observations are found in Sub- Section 3.2.2. 1. Growth of cells Both cell lines 1 and 2 underwent a 5 fold increase in fresh weight over the

254 growth cycle, as shown in Figures 3.2-3(c) of Sub-Section 3.2.2 and 3.2-4(c) of Sub- Section 3.2.2 respectively.

2. Production of methylated coumarins (i) Cell lines 1 (Figure 3.2-3(a), Sub-Section 3.2.2) and 2 (Figure 3.2-4(a), Sub-Section 3.2.2), exhibited similar abilities to methylate the hydroxylated coumarins aesculetin and fraxetin and consequently produced similar levels of methylated coumarins scopoletin and isofraxidin . (ii) The pattern of scopoletin production was similar in cell lines 1 and 2. The pattern of isofraxidin production was also similar in both cell lines. (iii) In both cell lines 1 and 2, the highest levels of scopoletin occurred in the lag phase with a smaller peak of activity occurring towards the end of the growth cycle. Isofraxidin levels on the other hand, increased overall throughout the growth cycle.

3. CMT Activity In cell line 1, fraxetin méthylation was very prominent during the growth cycle (Figure 3.2-3(b), Sub-Section 3.2.2), whilst aesculetin méthylation was low in comparison. In cell line 2 (Figure 3.2-4(b), Sub-Section 3.2.2) however, aesculetin méthylation was more prominent than fraxetin méthylation.

4.2.2 The Divergence in Methylating Abilities of Cell Lines 1 and 2 of A. altissima: Coumarin Méthylation In the méthylation of hydroxylated coumarins by the CMT enzyme of A. altissima cell suspension cultures, certain characteristic features were demonstrated. A detailed investigation of these features and their determinants were beyond the scope of this project. However, factors which could play possible roles in determining these features, have been discussed as follows:

1. In cell lines 1 (Figure 3.2-3, Sub-Section 3.2.2) and 2 (Figure 3.2-4, Sub-Section 3.2.2), two peaks of scopoletin production were observed at the earlier and latter parts

255 of the growth cycles respectively. The second peak of scopoletin production (which occurred towards the end of the growth cycle) is thought to be due to the 5 fold increase in cell weight of the cells, which coincided with this peak. The first peak of scopoletin production however may be as a response to stress, e.g. in response to the transfer of the cells unto fresh medium.

2. Fraxetin and aesculetin methyltransferase activities in cell lines 1 and 2 respectively, were similar to 1-HMT activity. However in spite of these high levels of CMT activity, levels of alkaloid produced by both cell lines were a 100 fold greater than levels of methylated coumarins isofraxidin and scopoletin. This observation may be due to 1 of 2 possible factors: a) The rapid metabolic turnover of methylated coumarins, isofraxidin and scopoletin. b) A block in the earlier part of the pathway leading to coumarin production i.e. the formation of aesculetin.

3. Cell lines 1 and 2 produced similar levels of the methylated coumarins scopoletin and isofraxidin . However, Fraxetin méthylation by the CMT enzyme was more prominent in cell line 1 than aesculetin méthylation and in cell line 2 the reverse was true i.e. aesculetin méthylation was more prominent than fraxetin méthylation. These similarities in the levels of scopoletin and isofraxidin produced by both cell lines, in spite of the differences in CMT activity, indicate that other factors apart from CMT activity are probably responsible for regulating the ability of A. altissima to methylate hydroxylated coumarins. These factors are listed below, some of them have been discussed previously in Sub- Section 4.1.2.2, and so will not be discussed in detail now. a) The availability of hydroxylated substrates, aesculetin and fraxetin. Neither of these substrates were detected in cell cultures of A. altissima, either

256 because they were present at very low levels, or because they were rapidly methylated. b) The availability of SAM c) The SAH levels

Factors b and c have been discussed in detail in Sub-Section 4.1.2.2, with respect to alkaloid méthylation. These factors could also apply to coumarin méthylation in a similar manner.

4. The fact that cell lines 1 and 2 differed in their abilities to methylate 1- hydroxycanthin-6-one, yet had similar abilities to methylate hydroxylated coumarins aesculetin and fraxetin, suggests that: A possible competition for available SAM or competition for available hydroxylated substrates may be among the factors which determine the methylating abilities of these cultures.

4.2.3 Purification and Characterisation of CMT CMT was only partially purified by the purification procedure employed, which involved the use of the DEAE Cellulose column. CMT was separated from 1-HMT by this column, but was not further purified. Both aesculetin and fraxetin methyltransferase activities were detected in cell cultures of A. altissima. However the CMT enzyme was generally referred to as CMT rather than aesculetin methyltransferase (AMT) and fraxetin methyltransferase (FMT). Since further purification of CMT was not embarked upon, it is not certain whether CMT is one enzyme which methylates both hydroxylated substrates or if CMT consists of 2 different enzymes aesculetin methyltransferase (AMT) and fraxetin methyltransferase (FMT). Further purification of CMT would be necessary in order to make conclusive statements on the characteristics of CMT.

4.2.4 The Proposed Pathway for the Biosynthesis of Coumarins in A. altissima Experiments were carried out to investigate the ability of CMT from A.

257 altissima cell cultures to methylate the hydroxylated coumarins aesculetin (6,7- dihydroxy-coumarin) and fraxetin (7,8-dihydroxy-6-methoxycoumarin) (Sub-Section 3.2.2). Further experiments were also carried out to investigate the ability of CMT to methylate not only aesculetin and fraxetin, but also other hydroxylated substrates including: scopoletin, isoscopoletin, fraxidin and isofraxidin (Sub-Section 3.5.2). The results obtained from these studies, which have been summarised below, made it possible to propose a biosynthetic pathway for the formation of coumarins in A. altissima.

4.2.4.1 The Méthylation of Coumarin Substrates by the CMT Enzyme of A. altissima 1. Two sequences for coumarin méthylation, termed sequences 1 and 2 (Figure 3.5-1, Sub-Section 3.5.2), have been identified in cell suspension cultures of A. altissima. Sequences 1 and 2 involve the mono-methylation of aesculetin (6,7- dihydroxycoumarin) to scopoletin (6-methoxy-7-hydroxycoumarin) and the monomethylation of fraxetin (6-methoxy-7,8-dihydroxycoumarin) to isofraxidin (6,8- dimethoxy-7-hydroxycoumarin), respectively.

2. Experiments carried out to investigate the further méthylation of scopoletin (7- hydroxy-6-methoxycoumarin) and isofraxidin (6,8-dimethoxy-7-hydroxycoumarin), showed that neither of these substrates were methylated by the CMT enzyme. However their isomers, isoscopoletin (6-hydroxy-7-methoxycoumarin) and fraxidin (6,7-dimethoxy-8-hydroxycoumarin), which were not identified in A. altissima cell suspension cultures, were methylated by CMT to presumably form scoparone (6,7- dimethoxycoumarin) and 6,7,8-trimethoxycoumarin respectively. From these results certain deductions were made: a. Aesculetin is methylated by the CMT enzyme from cell cultures of A. altissima at position C-6, to form scopoletin, but not at position C-7, to form its isomer isoscopoletin.

258 b. Scopoletin is not further methylated by CMT at the C-7 position to form scoparone (6,7-dimethoxycoumarin). c. CMT had the ability to further methylate isoscopoletin (not found in A. altissima cell suspension cultures), at the C-6 position to form scoparone. d. Fraxetin is methylated by the CMT enzyme from cell cultures of A. altissima, at position C-8, to form isofraxidin , but not at position C-7, to form its isomer fraxidin . e. Isofraxidin is not further methylated by CMT, at the C-7 position to form 6,7,8- trimethoxycoumarin. f. CMT had the ability to further methylate fraxidin (not found in A. altissima cell suspension cultures), at the C-8 position to form 6,7,8-trimethoxycoumarin. g. These results indicate that CMT is only capable of carrying out méthylations on hydroxy groups at positions C-6 and C-8 of the coumarin skeleton, but not postition C-7.

4.2.4.2 The Biosynthesis of Isofraxidin in A. altissima The results and deductions made from the studies mentioned in Sub-Section 4.2.4.1, resulted in the proposal of a biosynthetic pathway for the formation of methylated coumarins in A. altissima. Previous tracer studies carried out by Brown et al. (1988); Dewick et al. (1985, 1990), on the biosynthesis of the coumarin puberulin (Figure 1-12, Sub-Section 1.2.4.2.5), suggests that both sequences 1 and 2 (Figure 3.5-1, Sub-Section 3.5.2), for the méthylation of aesculetin and fraxetin respectively, are linked. In the proposed biosynthesis of puberulin, aesculetin is methylated to scopoletin and scopoletin is converted to fraxetin by 8-hydroxylation. Fraxetin is then methylated to isofraxidin which is finally converted to puberulin (Brown et al., 1988; Dewick et al., 1985, 1990). The biosynthetic pathway proposed for the coumarins of A. altissima is based on the biosynthesis of puberulin. The proposed pathway, which is shown in Figure 4-1, includes the following steps: Aesculetin (6,7-dihydroxycoumarin) is methylated

259 at C-6, by the CMT enzyme to form scopoletin (6-methoxy-7-hydroxycoumarin), which is then hydroxylated at position C-8 by a hydroxylase to form fraxetin (6- methoxy-7,8-dihydroxycoumarin). Finally fraxetin is methylated at position C-8 to yield isofraxidin (6,8-dimethoxy-7-hydroxycoumarin) as shown in Figure 4-1.

FIGURE 4-1 The Biosynthesis of Isofraxidine from Aesculetin in Cell Suspension Cultures of A. altissima

HO " 0 ^ 0

A e s c u l e t i n

S c o p o l e t i n

CH-jQ

F r a x e t i n

Isofraxidin

260 4.3 Conclusions 1. Cell lines 1 and 2 of A. altissima cell suspension cultures, which were grown under the same conditions, varied in their abilities to methylate l-hydroxycanthin-6-one. Cell line 1 produced 1-methoxycanthin-6-one as the major constituent and canthin-6-one and 1 -hydroxycanthin-6-one as minor constituents. Cell line 2, produced canthin-6- one as the major constituent and l-methoxycanthine-6-one and l-hydroxycanthin-6-one as minor constituents. The possible regulatory roles of the following factors have been speculated upon: 1-HMT enzyme activity; levels of SAM, the methyl group donor; feed back inhibition by 1-hydroxycanthin-6-one and other intermediates of the biosynthetic pathway and inhibition by méthylation products, l-methoxycanthin-6-one and SAH. Further work would be necessary in order to verify any of these speculations.

2. 1-Hydroxycanthin-6-one methyltransferase enzyme (1-HMT) was extracted and separated from the other methyltransferase enzyme of A. altissima (Coumarin methyltransferase, CMT). 1 -HMT was subsequently purified to a single protein from A. altissima cell cultures and characterised. However contamination of 1-HMT by low molecular weight proteins during purification, resulted in a poor loading capacity of the column used in the final purification step and consequently, low yields of the pure enzyme. Most experiments were therefore carried out on the products of purification steps prior to the final purification step.

3. 1-HMT was characterised as a monomer of molecular weight 60,000 - 63,000. 1 -HMT did not require divalent metal cations (Mg^^ and Mn^^) for activity and was inhibited by Cu^^. Its activity was inhibited significantly by SH group inhibitors, lA and p-CMB. The enzyme also demonstrated a requirement for SH protecting reagents, indicating that reduced sulphydryl groups played an active role in enzyme activity. 1-HMT was inhibited by products of the méthylation reaction, 1- methoxycanthin-6-one and SAH. SAH strongly inhibited enzyme activity, with a competitive mode of action and a Kj of 4 - 5 pM. 1-HMT exhibited optimum activity

261 at a temperature of 35 °C, a pH of 7 - 7.5 and an incubation time of 45 minutes. 1- HMT was also stable for 24 days when stored at -20 °C in 30 % glycerol.

4. 1-HMT was produced in relatively high yields by cell suspension cultures of A. altissima, which also grew quite rapidly.

5. The usefulness of 1-HMT in carrying out general méthylation reactions could be evaluated by carrying out further experiments on the scope of action (specificity), and the mode of action of 1-HMT. This should be followed by carrying out experiments to investigate the possibility of expanding the scope of action of 1-HMT. These experiments are discussed further in Section 4.4.

6. Two sequences for the méthylation of coumarins were identified in cell suspension cultures of A. altissima', i.e. sequence 1 for the méthylation of aesculetin to scopoletin and sequence 2 for the méthylation of fraxetin to isofraxidin . Time course studies carried out during the growth cycles of cell lines 1 and 2 of A. altissima cell suspension cultures, showed that in both cell lines scopoletin as well as isofraxidin were produced.

7. The coumarin methyltransferase enzyme (CMT) responsible for the méthylation of aesculetin and fraxetin, was found to be present in cell lines 1 and 2 at levels commensurate with 1-HMT levels, despite the fact that canthin-6-one alkaloid production was a 100 fold greater than coumarin production. The availability of hydroxylated coumarin substrates or rapid metabolism of methylated products were speculated upon as possible reasons for this discrepancy. Further work would however have to be done to substantiate this.

8. The CMT enzyme was extracted, and separated from the 1-HMT enzyme. CMT was only partially purified from A. altissima cell suspension cultures.

262 9. CMT is thought to be an enzyme which methylates hydroxylated coumarins specifically at positions C-6 and C-8, but not position C-7. The partially purified CMT enzyme was found to carry out the méthylation of aesculetin (6,7 dihydroxycoumarin), specifically at position C-6 to form scopoletin (6- methoxy-7-hydroxycoumarin). CMT however failed to methylate scopoletin at position C-7 to form scoparone (6,7-dimethoxycoumarin), but methylated its isomer isoscopoletin (6-hydroxy-7-methoxycoumarin), which is not found in A. altissima cultures) at position C-6 to from scoparone. The partially purified CMT enzyme also methylated fraxetin (7,8-dihydroxy-6-methoxycoumarin), specifically at the C-8 position to form isofraxidin (6,8-dimethoxy-7-hydroxycoumarin). CMT however failed to methylate isofraxidin at position C-7 to form 6,7,8-trimethoxycoumarin, but methylated its isomer fraxidin (6,7-dimethoxy-8-hydroxycoumarin), at position C-8 to presumably form 6,7,8-trimethoxycoumarin.

10. The méthylation pattern exhibited by CMT suggests that it is consistent with the pathway proposed in literature (Brown et at., 1988; Dewick, 1985, 1990) for the biosynthesis of puberulin via aesculetin, scopoletin, fraxetin and isofraxidin^ . It is thought that a similar pathway for the coumarin méthylation (from aesculetin to isofraxidin ) also exists in A. altissima cell suspension cultures. This would suggest that sequences 1 and 2 for coumarin méthylation are linked together; with aesculetin (6,7-dihydroxycoumarin) undergoing méthylation at C-6 to form scopoletin (6-methoxy-7-hydroxycoumarin), which then undergoes hydroxylation at position C-8 to form fraxetin (6-methoxy-7,8-dihydroxycoumarin). Fraxetin is thought to be finally methylated at position C-8 to form isofraxidin (6,8-dimethoxy-7- hydroxycoumarin).

263 4.4 Future Work Research into the methylating ability of A. altissima cell cultures involves a wide range of investigations, all of which could not possibly be completely covered in the course of this project. Some of the significant areas which were not studied, constitute areas of interest for future research.

4.4.1 The Potential use of 1-HMT in the General Méthylation of Compounds The possibility of using 1 -HMT in méthylation reactions can only be realised, if further investigation of 1-HMT enzyme activity is carried out. These would involve the following:

1. The specificity of action of 1-HMT should be studied by investigating its ability to methylate other hydroxylated compounds. This would give an indication of the scope of action of the methyltransferase enzyme. The substrate specificity of an enzyme gives an indication of the preference it displays towards certain substrates (Bohinski, 1979 a). Most enzymes exhibit a relative specificity i.e. they have a wider range of activity though still exhibiting a limited preference for a small number of chemically related materials (Bohinski, 1979 a). The detailed study of an enzyme’s substrate specificity, is the study of the structural parameters which determine whether a molecule will form a complex with the enzyme. These include the following:

(a) The native structure of the enzyme. The first step of an enzyme catalysed reaction, is the formation of the Enzyme- Substrate complex. The complex contains a number of weak bonds which can be formed and broken easily. The orientation of the substrate with respect to the enzyme and hence the specificity of enzyme action are determined by these bonds (Bowman and Rand, 1980).

264 (b) The thermodynamics of the molecular interaction involved. In the bound state, both the substrate and the enzyme become distorted, with the bond angles, electron distribution and interatomic distances differing from the free molecule (Bowman and Rand, 1980). The effects of the changes in size and shape of the proteins, on both binding and reaction velocity can be studied by measuring binding constants of inhibitors, in which the structure is systematically varied and by altering the structure of the substrates (Metzler, 1977). (c) The rate of binding of enzyme (E) and substrate (S) as well as the rate at which the complex is broken down. As a result of the forces in the ES complex, an intermediate or transitional state is reached. This rearranges rapidly to form the product which is released from the enzyme and restores the active centre (Metzler, 1977). Km reflects the rate at which the enzyme-substrate complex is formed and V^ax» the rate at which the enzyme-substrate complex breaks down to form the product (Bohinski, 1979 a).

2. The specific mode of action involved in the combination of the enzyme and the substrate. No single all inclusive mechanism of action could account for the precise mode of operation of enzymes in the process of E + S —> ES —> E -i- P. Each enzyme exhibits a unique mode of action, although it is known that enzymes having a similar function, demonstrate similarities in molecular structure and chemical action. Attempting to solve the mechanism of action of an enzyme may prove difficult primarily because the mechanism is complex in nature, involving several aspects at the active sites that are highly coordinated. In spite of the individuality in their modes of action, all enzymes operate according to the general principle that there exists a highly ordered set of chemical interactions between the bound substrate and the R-groups (side chains) of the amino-acid residues found in the active sites (the site of attachment of the substrate to the enzyme) (Bohinski, 1979 a). Determination of the mode of action of an enzyme therefore involves an investigation of the nature of

265 interactions occurring between substrates and amino acid residues at the active centre for the enzyme (Bowman and Rand, 1980). As a result of the specificity of action exhibited by enzymes, biochemists early adopted a lock and key theory, which stated that the substrate must fit to an active site precisely for reaction to occur. The major limitation of the lock and key hypothesis is that it is only applicable to the absolute specificity of enzymes, but does not account for the relative specificity. This is because this theory implies that the entire conformation of the enzyme is rigid. It therefore suggests that the recognition of the substrate, requires a precise, one of a kind structural compatibility between the active sites on the enzyme and the substrate (Bohinski, 1979 a). This discovery led to the proposal of an alternative concept known as the hypothesis of induced fit, which suggests that the correct configuration for interaction, is initiated in the active centre, only when the substrate approaches it (Bowman and Rand, 1980). According to this conformation, producing the best orientation of the active site residues, explains the high degree of relative specificity exhibited by several enzymes, since different substrates of similar structure, could initiate the conformational changes in the active sites.

3. Results obtained from studies of the mode of action of the enzyme would determine, how the structure of the enzyme can be altered in order to make the binding sites more accommodating to a wider variety of substrates.

4.4.2 The Biosynthesis of Coumarins in A. altissima A biosynthetic pathway for the production of coumarins in A. altissima cell suspension cultures has been proposed. This pathway (shown in Figure 4-1), leads to the formation of isofraxidin from aesculetin via scopoletin and fraxetin. This pathway will however have to be further substantiated. This would involve the following steps:

1. Further purification of the CMT enzyme, to determine if it constitutes:

266 (i) a single protein which methylates the OH group of aesculetin and fraxetin specifically at position C-6 and C-8 respectively but not at C-7, or (ii) a mixture of 2 different enzymes; aesculetin methyltransferase, (AMT), which has the ability to methylate aesculetin at position C-6 but not C-7 and fraxetin methyltransferase, (FMT) which methylates fraxetin at C-8 but not C-7.

2. The isolation and characterisation of the hydroxylase which catalyses the 8- hydroxylation of scopoletin to form fraxetin. This would involve firstly investigating the ability of cell suspension cultures of A. altissima to carry out this hydroxylation reaction and subsequently the isolation and characterisation of the enzyme.

3. The full identification of all the intermediates and enzymes involved in the biosynthesis of isofraxidin from aesculetin in A. altissima cell cultures. This is neccessary in order to fully substantiate the occurrence of the proposed biosynthetic pathway. Presently the only unidentified intermediate is fraxetin. Aesculetin, the starting material has also not been identified. During time course studies of A. altissima cell suspension cultures, fraxetin and aesculetin levels were investigated, but they were not found in these cultures. It is possible that they both undergo rapid méthylation or they are present in such low levels that large scale extractions would be required in order to identify them.

4. An investigation of the earlier steps of coumarin biosynthesis i.e. (leading to the formation of aesculetin), in A. altissima. This would further confirm the validity of the proposed pathway. Romero et al. (1993), reported that the enzyme chorismate mutase was found to be active in cell cultures of A. altissima. Chorismate mutase catalyses the conversion of chorismate to prephenate in the shikimate/chorismate pathway (Figure 1-4, Sub-Section 1.2.4.1.1). Prephenate is converted to L-phenylalanine, via phenylpyruvic acid (Poulson et al., 1993). Phenylalanine is then converted to 4'-hydroxycinnamic acid (p-coumaric acid), the

267 precursor of coumarins, by the enzyme phenylalanine ammonia lyase, PAL. Further substantiation of the existence of this pathway would involve the isolation and characterisation of the enzymes catalysing the conversion of prephenate to 4'-hydroxycinnamic acid from A. altissima cell suspension cultures. These enzymes are: prephenate dehydratase and phenylalanine aminotransferase (as shown in Figure 1-8, Sub-Section 1.2.4.2.1) and PAL (as shown in Figures 1-9 and 1-10 of Sub-Section 1.2.4.2.2 and 1.2.4.2.3 respectively).

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Zenk, M.H. (1978). The impact of plant cell culture on industry. In Frontiers of Plant Tissue Culture, (ed) T.A. Thorpe, pp 1-13, International Association for Plant Tissue Culture, University of Calgary, Alberta.

Zenk, M.H., El-Shagi, H., Arens, H., Stockigt, J., Weiler, E.W. and Deus B. (1977). Formation of the Indole alkaloids, serpentine and ajmalicine in cell suspension cultures of Catharanthus. In Plant Tissue Culture and it’s Biotechnological Application, (eds) W. Barz, E. Reinhard and M.H. Zenk, pp. 27-43. Springer-Verlag, Berlin.

291 LIST OF PUBLICATIONS

1. Osoba, O.A. and Roberts, M.F. (1993). Purification and Properties of 1- hydroxycanthin-6-one S-adenosyl-L-methionine methyltransferase from Ailanthus altissima cell cultures. Phytochemistry, 32(3), 665-671.

2. Osoba, O.A. and Roberts, M.F. (1993). Methyltransferase activity in Ailanthus altissima cell suspension cultures. Plant Cell Reports (In Press).

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