CHARACTERISATION OF A NOVEL NON-HENE DIOXYGENASE Mary G. Wallis Thesis presented for the Degree of Doctor of Philosophy University of Edinburgh r ACKNOWLEDGEMENTS I owe a great deal to my supervisor Dr. S. K. Chapman who has been unceasing in his help and enthusiasm throughout this study. I am deeply indepted to Drs. H.C. Baxter, R.L. Baxter, A. Cooper (University of Glasgow), J.G. Farmer, L.A. Gilmore, G.A. Reid, Miss H. Grant and Mr. J. Millar for help and expertise. Special thanks to Dr. N.C. McClure (University of Wales) for generous donations of bacteria and the use of unpublished material. My thanks are also due to Mrs. L. Marouf for the hectic typing of this thesis. I am very grateful to the Science and Engineering Research Council and also to Mr. and Mrs. R.G. Wallis for financial assistance over the last few years. I acknowledge the University of Edinburgh for the use of their facilities. - Finally, I would like to thank my family and friends for tremendous support, encouragement and many moments of madness, in particular during the preparation of this manuscript. (i) For my family, especially Jack Wallis and Campbell Beattie (ii) Inspiration did not come easily to Mary: 'I thought and pondered - vainly. I felt that blank incapability of invention which is the greatest misery of authorship, when dull Nothing replies to our anxious invocations. "Have you thought of a story?" I was asked each morning, and each morning I was forded to reply with a mortifying negative'. But revelation was at hand. Maurice Hindle on Mary Shelley (iii) DECLARATION Except where specific reference is made to other sources, the work presented in this thesis is the original work of the author. It has not been submitted, in whole or in part, for any other degree. Some of the results have already been published. Mary G. Wallis (iv) ABSTRACT A purification procedure has been developed for a novel extradiol dioxygenase, designated as 3-methyl- catechol 2,3-dioxygenase. The enzyme which is expressed in Escherichia coli, was originally derived from a Pseudomonas putida strain able to grow on toluidine. 3-Methylcatechol 2,3-dioxygenase was purified to homogeneity as judged by sodium dodecyl sulphate-poly- acrylamide gel electrophoresis (SDS-PAGE). Physical and kinetic properties of the purified enzyme were investigated. The enzyme consists of a single subunit type of Mr = 33,500 ± 2,000 by SDS-PAGE. Gel filtration indicated a molecular weight, under non-denaturing conditions, of 120,000 ± 20,000 consistent with the native enzyme existing as a tetramer of identical subunits. The NH 2 -terminal sequence (35 residues) has been determined and shows 50% identity with other extradiol dioxygenases. The structural charaqterisation of 3-methylcatechol 2,3-dioxygenase at the primary, secondary and quaternary levels indicates that the enzyme is typical of the extradiol dioxygenases. Evidence from several experiments, such as metal removal and replacement, oxidation and reduction, indicates that Fe (II) is essential for enzymatic function with the oxidised, ferric, form being inactive. No requirement for other cofactors was detected. (v) Thermal inactivation experiments demonstrated the complete stability of the enzyme up to 45°C for prolonged periods, however, above this temperature the enzymic activity was seen to decline. The kinetics of 3-methylcatechol 2,3-dioxygenase were investigated using UV/visible spectrophotometry and oxygen electrode polarography. Measurements were made under both standard and modified conditions. Typical saturation kinetics were observed for catechol, 3-methylcatechol and 4-methylcatechol as substrates. Data were analysed to give values of Vmax and Km. The substrate specificity for this enzyme was somewhat different from that seen for other catechol 2,3-dioxygenases, with 3-methylcatechol being cleaved at the highest rate. The Km values for the organic substrates were all around 0.3 AM, the lowest found for any dioxygenase to date. The Km for dioxygen was determined to be < 10 6 M. The enzyme consumed one mole of oxygen per mole of substrate in all three cases. The dependence of enzyme activity on pH follows a classic bell-shaped curve with a pH optimum of about 7.5 and pKa values of 6.9 (± 0.1) and 8.7 (± 0.1). The possibility that the lower pKa value might be due to an active site histidine was investigated by attempted modification of this residue. The implications of these results are discussed in relation to the structure and function of the enzyme. (vi) UNITS AND ABBREVIATIONS Abbreviations of units are of a standard form; dalton units Da grain(s) g litre(s) 1 second(s) s Other unit abbreviations include; molar absorption coefficient M- 1 cm' first order rate constant Both the single and three letter codes have been used to denote the amino acid residues; Amino Acid Three Letter One Letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Giutamine Gin Q Giutamic Acid Glu E Glycine Gly G Histidine His H (vii) Three Letter One Letter Amino Acid Abbreviation Symbol Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Vaiine Val V The following nomenclature has been adopted in referring to the dioxygenases; Enzyme Abbreviation Catechol 1,2-dioxygenase C1,2D Catechol 2,3-dioxygenase C2,3D 1, 2-Dihydroxybiphenyl 2, 3-dioxygenase 1, 2DHB2, 3D 1, 2-Dihydroxynaphthalene 2, 3-dioxygenase 1, 2DHN2, 3D 3, 4-Dihydroxyphenylacetate 2, 3-dioxygenase 3, 4DHPA2, 3D Gentisate 1,2-dioxygenase G1,2D 3-Methylcatechol 2, 3-dioxygenase 3MC2, 3D Protocatechuate 3, 4-dioxygenase 4D Protocatechuate 4, 5-dioxygenase 5D Steroid 4,5-dioxygenase 54,5D (viii) The following abbreviations have been employed in referring to the bioluimics; Chelating Liciand Abbreviation acetylacetonate acac - nitrilotriacetic acid NTA N,N'-(3, 3'dipropylamine)bis(salicylidene- amine) saldpt N, N' -ethylenebis (sal icyl ideneamine) salen N,N'-1, 2-benzenebis(salicylideneamine) saloph Other miscellaneous abbreviations include; Michaelis constant Km limiting value for reaction rate Vmax nicotinamide adenine dinucleotide NAD nicotinamide adenine dinucleotide phosphate NADP pounds per square inch p.s.i. revolutions per minute r.p.m. tris (hydroxymethyl) aminomethane tris (ix) LIST OF CONTENTS Page No. CHAPTER 1 : INTRODUCTION 1.1 Historical Perspective 1 1.2 Distribution of Oxygenases 3 1.3 Physiological Significance of Oxygenases 4 1.4 The Classification of Oxygenases 8 1.5 Structural Studies of Both the Intradiol and Extradiol Catechol Dioxygenases 16 1.6 Dioxygenase Biomimics 25 1.7 Mechanistic Studies of the Catechol Dioxy- genases 28 1.8 References 39 CHAPTER 2 : ISOLATION AND PURIFICATION OF 3- NETHYLCATECHOL 2, 3-DIOXYGENASE FROM ESCHERICHIA COLI 2.1 Introduction 50 2.2 Strains, Media and Growth of Bacteria 50 2.3 Enzyme Isolation 52 2.3.1 Step .1 : Crude Extract 52 2.3.2 Step 2 : Ammonium Sulphate Fractionation 54 2.3.3 Step 3 : Ion Exchange Chromatography 54 2.3.4 Step 4 : Gel Filtration 57 (x) 2 • 3 'd) Pace No. 2.3.5 Isolation and Purification Summary 57 2.4 Discussion of Isolation Techniques Employed and Comparison with Other Dioxygenase Preparations 60 2.4.1 Cell Lysis 60 2.4.2 Nucleic Acid Treatment 63 2.4.3 Acetone-Phosphate Buffer 64 2.4.4 Ammonium Sulphate Fractionation 65 2.4.5 Dialysis 66 2.4.6 Ion Exchange Chromatography 66 2.4.7 Pressure Dialysis 66 2.4.8 Gel Filtration 67 2.5 Discussion of a New Dioxygenase Purification Protocol 68 2.6 References 70 CHAPTER 3 : PRELIMINARY CHARACTERISATION OF 3- NETHYLCATECHOL 2, 3-DIOXYGENASE 3.1 Introduction 72 3.2 Experimental 72 3.2.1 Subunit Molecular Weight Determination 72 3.2.2 Native Molecular Weight Determination 72 3.2.3 NH 2 -Terminal Amino Acid Sequence Analysis 74 3.2.4 Thermal Stability 74 (xi) CHAPTER 3 (Cont'd) Page No. 3.3 Results and Discussion 78 3.3.1 Molecular Weight Determination 78 3.3.2 Amino Acid Sequence Analysis 81 3.3.3 Thermal Stability 106 3.4 Summary of Physical Properties 109 3.5 References 112 CHAPTER 4 : FURTHER CHARACTERISATION OF 3- METHYLCATEHOL 2, 3-DIOXYGENASE 4.1 Introduction 116 4.2 Experimental 116 4.2.1 Spectrophotometric Determination 116 4.2.2 Fe(II) Removal to Prepare the 3MC2,3D Apo-protein and Reconstruction of the Holo-enzyme by Fe(II) Replacement 117 4.2.3 Inactivation of 3NC2,3D by Oxidation and Reactivation of the Inactivated Enzyme by Re-reduction 117 4.2.4 Metal for Metal Replacement 118 4.3 Results and Discussion 118 4.3.1 Spectrophotometric Determination 119 4.3.2 Fe(II) Removal and Replacement ii 4.3.3 3MC2,3D Oxidation and Reduction 125 - (xii) 4.3 (Cont'd) Page No. 4.3.4 Metal for Metal Replacement, Metal Ions as probes to Obtain Comparative Information 127 4.4 Summary 131 4.5 References 133 CHAPTER 5 : KINETIC CHARACTERISATION OF 3-METHYL- CATECHOL 2, 3-DIOXYGENASE 5.1 Introduction 136 5.2 Experimental 137 5.2.1 Kinetic Studies 137 5.2.2 Attempted Modification of Histidine 139 5.2.3 Oxygen Consumption Studies 143 5.3 Results and Discussion 144 5.3.1 Kinetic Studies 144 5.3.2 pH Dependence of Enzyme Kinetics 153 5.3.3 Attempted Modification of Histidine 159 5.3.4 Oxygen Consumption Studies 161 5.3.5 Mechanistic Implications 163 5.4 Summary 164 5.5 References 166 CHAPTER 6 : METHODS AND MATERIALS 6.1 Growth Media 172 6.1.1 Luria Broth (L.B.) for the Growth of E.