Expanding the Role of Oxidoreductases in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

Home , Benzylisoquinoline, Dihydrosanguinarine, Papaver setigerum, Protopine, Sanguinarine

University of Calgary PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2015-11-19 Expanding the Role of Oxidoreductases in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy

Farrow, Scott Cameron

Farrow, S. C. (2015). Expanding the Role of Oxidoreductases in Benzylisoquinoline Alkaloid Metabolism in Opium Poppy (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26041 http://hdl.handle.net/11023/2648 doctoral thesis

University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY

Expanding the Role of Oxidoreductases in Benzylisoquinoline Alkaloid Metabolism in Opium

Poppy

Scott Cameron Farrow

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

November, 2015

Benzylisoquinoline alkaloids (BIAs) are a large and structurally diverse group of plant specialized metabolites with several possessing pharmacological properties including the analgesic morphine, the cough suppressant codeine, and the vasodilator papaverine. In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by oxidoreductases belonging to the 2-oxoglutarate/Fe(II)-dependent dioxygenase gene family, namely thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). When assayed with a wider range of BIAs, CODM, T6ODM, and the functionally unassigned paralogs

DIOX2 and DIOX7, renamed protopine O-dealkylase (PODA) and papaverine 7-O-demethylase

(P7ODM), respectively, showed novel and efficient O-dealkylation activities, including regio and substrate-specific O-demethylation and O,O-demethylenation. Preferred substrates for O,O demethylenation by CODM and PODA were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing (VIGS) used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy. Furthermore, the efficient substrate- and regio-specific 7-O-demethylation of papaverine by P7ODM yielding pacodine suggests an unexpected biosynthetic route to pacodine. In addition to these findings, my thesis investigated oxidoreductases from the cytochromes P450 (CYP) and aldo-keto reductase (AKR) gene families. Using the functionally characterized codeinone reductase (COR) AKR translated nucleotide sequence as a query, we identified a COR paralogue from an opium poppy transcriptome database that was fused and in frame with a CYP. The resulting protein fusion

ii catalyzed the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The fusion protein, renamed reticuline epimerase (REPI), was detected in opium poppy and in Papaver bracteatum,

which accumulates the morphinan alkaloid thebaine. In contrast, orthologs encoding independent

CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2

dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan

alkaloids. Suppression of REPI transcripts using VIGS in opium poppy reduced levels of (R)

reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its

O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for

known steps of morphine biosynthesis.

iii Acknowledgements

There are several people that helped me during the past six years of my PhD, and without them this thesis would not have been possible. First and foremost, I would like to thank my supervisor Dr. Peter Facchini for providing me with the opportunity to work on these exciting projects. Your guidance and support have been invaluable over the duration of my PhD.

I would like to thank my supervisory committee and several faculty members for their support and encouragement throughout my studies. These include: Dr. Dae-Kyun Ro, Dr.

Marcus Samuel, Dr. Doug Muench and Dr. C.C. Chinnappa. I would also like to thank Dr. Chris

Schofield and Dr. Justin MacCallum for participating in my defence.

Much of my success during my PhD would not have been possible without the assistance of many colleagues. In particular, Dr. Darcy Burns for his assistance with NMR, Dr. Isabel

Desgané-Penix for her discussions about plant physiology, Dr. Jill Hagel for her tutelage on dioxygenases and biochemical techniques, Gina Ro for her guidance with yeast work, Dr.

Andrew Stopford for his assistance with mass spectrometry, and Dr. Shaobo Wu for his assistance with molecular biology techniques.

I would also like to thank all of my labmates for their comraderie and teamwork. This included Dr. G. Beaudoin, Crystal Bross, Dr. T. Dang, Donald Dinsmore, Ryan Groves, Jeremy

Morris, A.K. Onoyovwi and Dr. C. Wijekoon.

I am also grateful for the support I received from Alberta Innovates Technology Futures and the Natural Sciences and Engineering Research Council of Canada.

I am honoured to have such great friends who have helped to keep me grounded and motivated throughout my PhD. In particular, I owe a big thank you to Dr. Glen Uhrig, Dr. Neil

iv Emery, Donald Dinsmore, Dave Bak, Bill Cocks, Dr. Tom Fudalewski, Dr. Moonhyuk Kwon,

Dr. Matthew White, Adam Bendall, and Dwayne Scott.

Of course, none of this would have been possible without my family. Grandma Jessie,

Grandma Audrey, Grandpa Jack, Grandpa Wilf, Mom Jan, Dad Kent, Mom Mary, Dad Terry, sister Erin, brother Cory, sister Melanie, sister Kelly, sister Marcia, nephew Jake, niece Keira, nephew Wynton, nephew Ruben, nephew Bowen, uncle Wilson, uncle Big Jeff, uncle Little Jeff,

Aunts, Uncles…Thank you for your endless support and encouragement.

Most importantly, to my wife Gillian, I could not have done any of this without you.

Your support, encouragement and unshakable foundation are the source of my strength. You got me through the tough times and celebrated with me through the great times. Our team approach to accomplishing goals is one of our biggest strengths. I look forward to what the future holds for our growing family.

Dedication

To Gillian and Leona

vi Table of Contents

Abstract ...... ii Acknowledgements ...... iv Dedication ...... vi Table of Contents ...... vii List of Tables ...... xi List of Figures and Illustrations ...... xii Publications and Patents ...... xv List of Symbols, Abbreviations and Nomenclature ...... xvii Epigraph ...... xix

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Opium Poppy ...... 1 1.2 Benzylisoquinoline Alkaloids ...... 2 1.2.1 Occurrence, Function and Medicinal Relevance of BIAs ...... 2 1.2.2 BIAs of Opium Poppy ...... 4 1.3 Biosynthesis of Benzylisoquinoline Alkaloids ...... 4 1.4 Aldo-Keto Reductases ...... 7 1.4.1 General Features of AKRs ...... 7 1.4.2 AKRs in Plant Metabolism ...... 8 1.4.3 AKRs in BIA Metabolism ...... 8 1.5 Cytochromes P450 Monoxygenases ...... 10 1.5.1 General Features of CYPs ...... 10 1.5.2 CYPs in Plant Metabolism ...... 13 1.5.3 CYPs in Secondary Metabolism ...... 15 1.5.4 CYPs in BIA Metabolism ...... 15 1.5.5 The Plant CYP Goldmine ...... 20 1.6 2-oxoglutarate/Fe(II)-dependent Dioxygenases ...... 20 1.6.1 General Features of ODDs ...... 20 1.6.2 Structural and Mechanistic Features of 2-oxoglutarate/Fe(II)-dependent Dioxygenases...... 22 1.6.3 ODDs in Primary Metabolic Networks ...... 24 1.6.4 ODDs in Plant Specialized Metabolism ...... 24 1.6.5 ODDs in Benzylisoquinoline Alkaloid Biosynthesis ...... 25 1.7 Gene Fusions ...... 26 1.8 Strategies for Gene Discovery in Plant Specialized Metabolism ...... 27 1.8.1 Traditional Approaches ...... 27 1.8.2 Omics Strategies ...... 28 1.8.3 Virus-Induced Gene Silencing ...... 29 1.9 Objectives ...... 30

CHAPTER TWO: MATERIALS AND METHODS ...... 32 2.1 Plant Material ...... 32 2.2 Chemicals and Reagents ...... 32 2.3 Benzylisoquinoline Alkaloids ...... 33 2.3.1 Commercially Available BIAs ...... 33

vii 2.3.2 Opium Derived BIAs ...... 33 2.3.3 Enzymatically Prepared BIAs ...... 33 2.4 Solid Phase Extraction ...... 35 2.4.1 (S)-N-methylcoclaurine ...... 35 2.4.2 α-Hydroxyreticulines ...... 35 2.4.3 Pacodine ...... 36 2.5 Semi-Preparative HPLC of Pacodine and α-Hydroxyreticulines ...... 36 2.6 Gene Isolation, Expression Vector Construction, Recombinant Gene Expression and Protein Purification ...... 37 2.6.1 Chapter 3: Identification and Cloning of ODDs ...... 37 2.6.2 Chapter 3: Recombinant Protein Production ...... 37 2.6.3 Chapter 3: Protein Purification ...... 38 2.6.4 Chapter 4: Identification and Cloning of DIOX7 ...... 40 2.6.5 Chapter 4: Recombinant Protein Production ...... 40 2.6.6 Chapter 4: Protein Purification ...... 40 2.6.7 Chapter 5: Identification and Cloning of AKRs and CYPs ...... 41 2.6.8 Chapter 5: Recombinant Protein Production in Sacchoromyces cereviseae ...42 2.6.9 Chapter 5: Recombinant Protein Production in Escherichia coli ...... 43 2.7 Enzyme Assays ...... 46 2.7.1 Chapter 3 ...... 46 2.7.2 Chapter 4 ...... 46 2.7.3 Chapter 5: Reductase Assays ...... 47 2.7.4 Chapter 5: Cytochrome P450 Assays ...... 47 2.8 Analysis of Enzyme Assays ...... 48 2.8.1 Chapter 3 ...... 48 2.8.2 Chapter 4 ...... 49 2.8.3 Chapter 5 ...... 49 2.9 Reaction Product Identification ...... 50 2.9.1 Triple Quadrupole Mass Spectrometry Analysis ...... 50 2.9.2 LTQ-Orbitrap XL Analysis ...... 51 2.9.3 NMR of α-hydroxyreticulines ...... 51 2.9.4 NMR of Pacodine ...... 52 2.10 NMR Processing ...... 53 2.11 Reaction Mechanism Assays ...... 54 2.11.1 Nash Assay ...... 54 2.11.2 Formate Dehydrogenase Assay ...... 54 2.12 Virus-induced Gene Silencing ...... 55 2.12.1 Vector Construction and Methodology ...... 55 2.12.2 Real Time Quantitative PCR ...... 56 2.12.3 Alkaloid Extraction ...... 57 2.12.4 VIGS Analysis ...... 57 2.12.5 Chapter 5: (S) and (R)-Reticuline Percentage...... 58 2.13 LC-MS Analysis of Papaverine and Pacodine ...... 59 2.14 Phylogenetic Analysis...... 59 2.15 Genbank Accession Numbers ...... 60 2.15.1 Accession Numbers of ODDs Sequences Used in this Thesis ...... 60

viii 2.15.2 Accession Numbers of AKR Sequences Used in this Thesis ...... 61 2.15.3 Accession Numbers of CYP Sequences Used in this Thesis ...... 61 2.16 Gene Expression Analysis in Different Plant Organs ...... 62

CHAPTER THREE: DIOXYGENASES CATALYZE O-DEMETHYLATION AND O,O DEMETHYLENATION WITH WIDESPREAD ROLES IN BENZYLISOQUINOLINE ALKALOID METABOLISM IN OPIUM POPPY .....63 3.1 Introduction ...... 63 3.2 Results ...... 68 3.2.1 Phylogenetic Analysis of Opium Poppy ODDs ...... 68 3.2.2 Biochemical Characterization of Opium Poppy ODDs ...... 70 3.2.3 Substrate Range ...... 78 3.2.4 Reaction Kinetics ...... 79 3.2.5 Reaction Mechanics ...... 81 3.2.6 Virus-Induced Gene Silencing ...... 83 3.3 Discussion ...... 91

CHAPTER FOUR: PAPAVERINE 7-O-DEMETHYLASE, A NOVEL 2 OXOGLUTARATE/FE(II)-DEPENDENT DIOXYGENASE FROM OPIUM POPPY ...... 103 4.1 Introduction ...... 103 4.2 Results ...... 107 4.2.1 Isolation of DIOX7 ...... 107 4.2.2 Expression and Characterization of DIOX7 ...... 108 4.2.3 Biochemical Properties of P7ODM ...... 111 4.2.4 P7ODM Transcript Levels in Different Opium Poppy Organs ...... 113 4.2.5 Pacodine and Papaverine Levels in Different Opium Poppy Organs ...... 113 4.3 Discussion ...... 115

CHAPTER FIVE: STEREOCHEMICAL INVERSION OF (S)-RETICULINE BY A CYTOCHROME P450 FUSION IN OPIUM POPPY ...... 118 5.1 Introduction ...... 118 5.2 Results ...... 123 5.2.1 COR Paralog Search Identifies CYP82Y2 in Opium Poppy ...... 123 5.2.2 CYP82Y2 is Reticuline Epimerase ...... 127 5.2.3 Requirement for N-methylated Substrates ...... 134 5.2.4 Physiological Characterization of Reticuline Epimerase ...... 140 5.3 Discussion ...... 146

CHAPTER SIX: OVERVIEW, FUTURE PERSPECTIVES AND CONCLUSIONS ...150 6.1 Overview of Thesis Research ...... 150 6.1.1 2-oxoglutarate/Fe(II)-dependent Dioxygenases in BIA Metabolism ...... 150 6.1.2 Stereochemical Inversion of Reticuline by a P450 Oxidoreductaase Fusion Protein in Opium Poppy...... 151 6.2 Future Perspectives ...... 152 6.2.1 ODD O-dealkylases from Other Plant Species ...... 152 6.2.2 Potential Significance of Protein Fusions ...... 153

ix 6.2.3 Characterization of Novel BIA Biosynthetic Genes ...... 154 6.2.4 Regulation of BIA biosynthesis ...... 155 6.2.5 Reconstitution of BIA Biosynthetic Pathways in Microorganisms...... 155 6.2.6 Creating a Deeper Specialized Metabolite Database ...... 157 6.3 Conclusion ...... 158 Appendix ...... 179

x List of Tables

Table 3.1. Kinetic values for PODA and CODM with cryptopine and (S)-scoulerine, respectively ...... 102

Table 5.1. Kinetic data for PsREPI, PsDRS, PrDRS, PsDRR1 and PrDRR ...... 133

xi List of Figures and Illustrations

Figure 1.1. Medicinally important BIAs ...... 3

Figure 1.2. Biogenesis Of BIA subgroups...... 6

Figure 1.3. CYP and AKR naming convention ...... 7

Figure 1.4. Biosynthesis of morphine in opium poppy ...... 9

Figure 1.5. Important CYP structures ...... 12

Figure 1.6. CYP catalytic cycle ...... 14

Figure 1.7. BIA reactions catalyzed by CYP80 family members ...... 16

Figure 1.8. CYP719 reactions in BIA metabolism ...... 18

Figure 1.9. CYP82 family members involved in BIA metabolism...... 19

Figure 1.10. Sequence alignments of O-demethylases from Papaver somniferum and ODDs from other plants for which crystal structures have been determined ...... 23

Figure 1.11. Proposed mechanisms of 2-oxoglutarate binding and formation of the reactive iron-oxo intermediate for the oxidation of the prime substrate ...... 24

Figure 2.1. SDS-PAGE of recombinant opium poppy ODD proteins produced in Escherichia coli ...... 39

Figure 2.2. SDS-PAGE and immunoblot analysis of purified recombinant P7ODM produced in Escherichia coli ...... 41

Figure 2.3. Heterologous production and/or purification of recombinant AKR and CYP enzymes ...... 45

Figure 3.1. Biosynthesis of major protopine alkaloids and sanguinarine in opium poppy ...... 67

Figure 3.2. Rooted neighbor-joining phylogenetic tree for opium poppy ODDs and others reported from plants ...... 69

Figure 3.3. LC-MS extracted ion chromatograms of enzyme assays using CODM, T6ODM, or PODA with protopine alkaloids ...... 73

Figure 3.4. Relative activity, reaction catalyzed, and regiospecificity of T6ODM, CODM, and PODA with different benzylisoquinoline alkaloids as substrates ...... 75

Figure 3.5. Identification of ODD reaction products using allocryptopine as the substrate by collision-induced dissociation mass spectrometry ...... 77

xii Figure 3.6. Steady-state enzyme kinetics of purified recombinant (A) PODA and (B) CODM using cryptopine and (S)-scoulerine, respectively, as substrates ...... 80

Figure 3.7. Formaldehyde is a byproduct of the O-demethylation, but not the O,O demethylenation of benzylisoquinoline alkaloids ...... 82

Figure 3.8. Virus-induced gene silencing of T60DM, CODM, and PODA in opium poppy. (A) Detection of viral coat protein transcripts by RT-PCR (B) Relative CODM and T60DM transcript level...... 86

Figure 3.9. Effect of virus-induced gene silencing on the accumulation of selected BIAs using pTRV2 constructs designed to suppress the transcript levels in opium poppy of all ODDs, T6ODM, or CODM compared with empty vector controls...... 88

Figure 3.10. Effect of virus-induced gene silencing on the accumulation of selected BIAs using pTRV2 constructs designed to suppress the transcript levels in opium poppy of all ODDs, T6ODM, or CODM compared with empty vector controls...... 90

Figure 4.1. Proposed biosynthesis of papaverine, pacodine and palaudine from (S) norcoclaurine in opium poppy ...... 106

Figure 4.2. SDS-PAGE and immunoblot analysis of purified recombinant P7ODM produced in Escherichia coli ...... 107

Figure 4.3. O-demethylation of papaverine by P7ODM confirmed using LC-MS ...... 109

Figure 4.4. Benzylisoquinoline alkaloids tested as potential substrates of P7ODM ...... 110

Figure 4.5. In vitro characterization of affinity-purified P7ODM ...... 112

Figure 4.6. Relative abundance of P7ODM transcripts in opium poppy ...... 114

Figure 5.1. Complete morphine biosynthetic pathway in opium poppy ...... 121

Figure 5.3. Maps of cDNAs and genes encoding reticuline epimerase (REPI), 1,2 dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reducatse (DRR) ...... 124

Figure 5.4. Unrooted phylogenetic trees for selected (A) cytochrome P450 monoxygenases in the CYP82 family and (B) NADPH-dependent aldo-keto reductases ...... 126

xiii Figure 5.5. Heterologous production and/or purification of recombinant enzymes ...... 129

Figure 5.6. Catalytic functions of PsREPI, PsDRS and PrDRS using (S)-reticuline, 1,2 dehydroreticuline, and (R)-reticuline as substrates ...... 130

Figure 5.7. Catalytic functions of PsDRR1 and PrDRR using (S)-reticuline, 1,2 dehydroreticuline, and (R)-reticuline as potential substrates ...... 131

Figure 5.8. Chiral separation and detection by HPLC-UV showed the formation of (R) reticuline by PsDRR1 and PrDRR from 1,2-dehydroreticuline ...... 132

Figure 5.9. PsAKR2 does not show the same function as PsDRR1...... 133

Figure 5.10. Benzylisoquinoline alkaloids tested as potential substrates of 1,2 dehydroreticuline synthase ...... 135

Figure 5.11. Catalytic functions of PsREPI, PsDRS and PrDRS on (S)-N-methylcoclaurine as a substrate ...... 136

Figure 5.12. pH optima of PsREPI, PsDRS, PsDRR1, PrDRS and PrDRR ...... 137

Figure 5.13. Steady-state enzyme kinetics for PsREPI, PsDRS, PrDRS, PsDRR1 and PrDRR. 138

Figure 5.14. Function of PsDRR1 and PrDRR on contaminants in the authentic 1,2 dehydroreticuline standard ...... 139

Figure 5.15. Virus-induced gene silencing in opium poppy supports the role of PsREPI in morphinan alkaloid biosynthesis ...... 142

Figure 5.17. Relative transcript abundance of benzylisoquinoline alkaloid biosynthetic genes in different opium poppy organs ...... 145

Figure 6.1. Examples of BIAs with altered O-methylation patterns (right) relative to the established or putative precursors (left) ...... 153

xiv Publications and Patents

All of the work described in this thesis has been published in peer-reviewed journals and patents, as presented below. The contributions of other authors on these publications are indicated at the beginning of each chapter where applicable.

Publications

Chapter 1 included sections from:

Farrow, S.C. and Facchini, P.J. (2014) Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism. Frontiers in Plant Science and Chemodiversity, 5, 1– 15.

Chapter 2 included methodologies from:

Farrow S.C. and Facchini P.J. (2015) Papaverine 7-O-demethylase, a novel 2 oxoglutarate/Fe(II)-dependent dioxygenase from opium poppy. FEBS Letters, 589, 2701– 2706.

Farrow S.C., Hagel, J.M., Beaudoin, G.A.W., Burns, D.C. and Facchini P.J. (2015) Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Biol., 11, 728–732.

Farrow S.C. and P.J. Facchini. (2013) Dioxygenases catalyze O-demethylation and O,O demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy. JBC, 288, 28997–29012.

Chapter 3 was adapted from:

Chapter 4 was adapted from:

Farrow S.C. and P.J. Facchini. (2015) Papaverine 7-O-demethylase, a novel 2 oxoglutarate/Fe(II)-dependent dioxygenase from opium poppy. FEBS Letters. 589, 2701– 2706.

xv Chapter 5 was adapted from:

Farrow S.C., Hagel, J.M., Beaudoin, G.A.W., Burns, D.C. and Facchini, P.J. (2015) Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Bio., 11, 728–732.

Publications not Included in this Thesis:

Hagel, J., Morris, J., Lee, E-J., Desgagné-Penix, I., Bross, C., Chang, L., Chen, X., Farrow, S.C., Zhang., Y., Soh, J., Sensen, C. and Facchini, P.J. (2015) Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants. BMC plant Biology. 15, 1–16.

Desgagne-Penix, I., Farrow, S.C., Cram, D., Nowak, J. and Facchini, P.J. (2012) Integration of deep transcript and targeted metabolite profiling for eight cultivars of opium poppy. Plant Molecular Biology, 79, 295–313.

Farrow, S.C., Hagel, J.M., Facchini, P.J. (2012) Transcript and metabolite profiling of 18 plant species that produce benzylisoquinoline alkaloids. Phytochemistry, 77, 79–88.

Dang, T.T., Onoyovwi, A., Farrow, S.C. and Facchini, P.J. (2012) Biochemical genomics for gene discovery in benzylisoquinoline alkaloid biosynthesis in opium poppy and related species. Methods in Enzymology, 515, 231–266.

Patents

Facchini, P.J., Farrow, S.C. and Beaudoin, G.A.W. Compositions and methods for making (R) reticuline. United States Patent Application (61/911,759) issued December 4, 2013.

Facchini, P.J., Hagel, J.M., Martin, V., Ekins, A., Fossati, E., Lauzon, J.F. and Farrow, S.C. Thebaine 6-O-demethylase and codeine O-demethylase from Papaver somniferum. Patent Cooperation Treaty Application (UNTI.P0096WO) issued November 12, 2010.

xvi List of Symbols, Abbreviations and Nomenclature

2OG 2-oxoglutarate ODD 2-oxoglutarate/Fe(II)-dependent dioxygenase 4HPAA 4-Hydroxyphenylacetaldehyde 4’OMT (S)-3’-hydroxy-N-methylcoclaurine 4’-O-methyltransterase 6OMT (S)-Norcoclaurine 6-O-methyltransferase 7OMT (R,S)-reticuline-7-O-methyltransferase ACCO 1-aminocyclopropane-1-carboxylic acid oxidase AKR Aldo-keto reductase BBE Berberine bridge enzyme BIA Benzylisoquinoline alkaloid BLAST Basic local alignment search tool CID Collision-induced dissociation CjNCS Coptis japonica norcoclaurine synthase CNMT (R,S)-coclaurine N-methyltransferase CODM Codeine O-demethylase CP Coat protein CPR Cytochrome P450 reductase CYP Cytochrome P450 DRS Dehydroreticuline synthase DRR Dehydroreticuline reductase ELF-1a Elongation factor 1-alpha EST Expressed sequence tag ESI Electrospray ionization GAPDH Glyceraldehyde 3-phosphate dehydrogenase LC-MS Liquid chromatography-tandem mass spectrometry LDOX Leucoanthocyanidin synthase LTQ-Orbitrap Linear Trap Quadrupole Orbitrap M-MLV-RT Murine leukemia virus reverse transcriptase M Multiple peaks (NMR) MCS Multiple cloning site MRM Multiple reaction monitoring MSH N-methylstylopine 14-hydroxylase NaAsc Sodium ascorbate NADP+ Nicotinamide adenine dinucleotide phosphate oxidized form NADPH Nicotinamide adenine dinucleotide phosphate reduced form N7OMT Norreticuline 7-O-methyltransferase NCS (S)-Norcoclaurine synthase NMCH (S)-N-methylcoclaurine 3’-hydroxylase NMR Nuclear magnetic resonance NMT N-methyltransferase OMT O-methyltransferase ORF Open reading frame P7ODM papaverine 7-O-demethylase Polyubiquiton-10 UBQ10

xvii PODA protopine O-dealkylase PCR Polymerase chain reaction qRT-PCR Quantitative real-time polymerase chain reaction T6ODM Thebaine 6-O-demethylase TNMT Tetrahydroprotoberberine N-methyltransferase P6H Protopine 6-hydroxylase PCR Polymerase chain reaction Pr Papaver rhoeas Ps Papaver somniferum PTGS Post-transcriptional gene silencing REPI Reticuline epimerase RT-PCR Reverse transcriptase polymerase chain reaction S Singlet peak (NMR) SDR Short-chain dehydrogenase/reductase SOMT Scoulerine 9-O-methyltransferase TRV Tobacco rattle virus VIGS Virus-induced gene silencing UTR Un-translated region

xviii

Epigraph

“For me, it is far better to grasp the Universe as it really is than to persist in delusion, however satisfying and reassuring” -Carl Sagan

xix Chapter One: Introduction

1.1 Opium Poppy

Opium poppy (Papaver somniferum) has been used as a source of medicine by humankind for millennia. Even Neolithic humans cultivated opium poppy for its medicinal properties, and it is likely that today’s version of opium poppy is the result of centuries of breeding (Bernáth,

1999); a situation that is analogous to modern day cereal crops (Unterlinner et al., 1999). The significance of opium poppy through the ages is underscored by its mention in ancient Sumerian tablets (3000 BCE), as a theme in ancient artwork such as the Minoan ‘poppy goddess’ (1400–

1100 BCE), and its place in early literary works like Homer’s Iliad (Bernáth, 1999). Since its roots in prehistoric cultures, opium poppy has emerged today as one of the most important plants in the history of pharmaceuticals owing to its unique ability among plants to produce the potent benzylisoquinoline alkaloid (BIA) analgesic morphine. In fact, opium poppy remains the sole commercial source of morphine owing to a chemical synthesis that is commercially not competitive. In addition to morphine, opium poppy produces a variety of other pharmacologically important BIAs including the cough suppressant and analgesic codeine, the vasodilator papaverine, and the antitussive and promising anticancer drug noscapine (Hagel and

Facchini, 2013). In contrast to its importance for the treatment of pain and other human ailments, opium poppy has been the subject of considerable controversy throughout human history owing to its role in international conflicts like the Anglo-Chinese opium wars and the opiate drug epidemic (Hagel and Facchini, 2013). As such, a delicate balance is maintained between access to- and regulation of opium poppy.

1 1.2 Benzylisoquinoline Alkaloids

1.2.1 Occurrence, Function and Medicinal Relevance of BIAs

BIAs are a large and structurally diverse group of plant specialized metabolites with

approximately 2,500 elucidated structures that are distributed widely among the flowering plants

with the greatest concentration occurring in members of the Ranunculales (Ziegler and Facchini,

2008). Within the Ranunculales, an impressive array of BIA structural diversity is seen

throughout the plant families Papaveraceae, Ranunculaceae, Berberidaceae, and

Menispermaceae. Whereas some BIAs are found throughout numerous species (e.g.

protoberberines), others (e.g. benzo[c]phenanthridines) are taxonomically restricted, and some

(e.g. morphine) are isolated to one species (Ziegler and Facchini, 2008; Hagel and Facchini,

2013).

Unlike primary metabolites, BIAs are not essential for the normal growth and development of the plant but, rather, enhance its fitness based on their purported roles as defense compounds

(Wink, 2003; Weiss et al., 2006). Paradoxically, much more is known about the anthropogenic uses of BIAs than their ecophysiological roles, and in this context, a plethora of humankind’s oldest and new medicines are derived or inspired from those found in plants. Notable examples include the aforementioned morphine, codeine, papaverine, and noscapine, but also the antimicrobials sanguinarine and berberine (Stermitz et al., 2000; Hagel and Facchini, 2013), the

muscle relaxant tubocurarine (Thompson, 1980), the antitussive glaucine (Cortijo et al., 1999),

and the semi-synthetic antiparkinson agent apomorphine (Figure 1.1; Millan et al., 2002).

Considering the number of BIAs in nature, there are likely several more with unknown

pharmacological activities.

Figure 1.1. Medicinally important BIAs. Notable pharmacological activities of BIAs include analgesic, antitussive, anticancer, antimicrobial, antiparkinson and as vasodilators.

3 1.2.2 BIAs of Opium Poppy

It has been said that opium poppy produces up to 40 BIAs (Dewick, 2009), however, when considering pathway intermediates and minor BIA constituents, the number is closer to 100. The most prominent of these belong to the morphinan, phthalide, 1-benzylisoqunoline, benzo[c]phenanthridine and aporphine BIA classes, while representatives of the rhoeadine, protopine and protoberberine sub-types are found at lower concentrations (Bernáth, 1999;

Desgagné-Penix et al., 2012) . This general catalogue, however, can fluctuate according to the opium poppy cultivar, which often displays a characteristic chemotype. Not surprisingly, several chemotypes have been bred to contain high concentrations of narcotic alkaloids such as morphine (Chauhan et al., 1987; Desgagné-Penix et al., 2012), whereas others contain high

concentrations of noscapine (Frick et al., 2005), or reticuline (Allen et al., 2004).

1.3 Biosynthesis of Benzylisoquinoline Alkaloids

Early pathway schemes (Winterstein and Trier, 1910; Pictet and Spengler, 1911; Robinson,

1917) and radiolabeled tracer studies (Stadler et al., 1987; Stadler et al., 1989; Stadler and Zenk,

1990) were paramount for establishing the common biosynthetic route to BIAs (Hagel and

Facchini, 2013), whereby two molecules of the amino acid L-tyrosine undergo decarboxylation

and meta-hydroxylation or transamination and decarboxylation to yield dopamine and 4

hydroxyphenylacetaldehyde (4HPAA), respectively (Beaudoin and Facchini, 2014). The stereo-

selective condensation of these two tyrosine derivatives by the characteristic BIA enzyme (S)

norcoclaurine synthase (Rueffer et al., 1981; Samanani et al., 2004) is the committed step in BIA

biosynthesis and yields the first BIA (S)-norcoclaurine. The 1-benzylisoquinoine scaffold of (S)

norcoclaurine or related derivatives such as the central branch-point intermediate (S)-reticuline,

serve as the foundation for all other BIA structural types, which are formed through defining C-C

or C-O phenol coupling reactions catalyzed by flavoprotein oxidase and cytochromes P450

4 monoxygenases (CYPs) (Figure 1.2; Hagel and Facchini, 2013). Remarkably, the vast array of

BIA structures contained within each structural category can be explained through functionalization of their ring system by a relatively restricted number of enzyme types including

S-adenosyl-L-methionine-dependent N- and O-methyltransferases, flavoprotein oxidases, carboxyl esterases, acetyl-transferases, CYPs, short-chain reductases, aldo-keto reductases

(AKRs), and 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODDs). My thesis focused on members of the CYP, AKR and ODD oxidoreductases. The following sections introduce these enzymes.

Figure 1.2. Biogenesis of BIA subgroups. Several BIA structural types can be understood through the oxidative C-C or C-O phenol coupling of 1-benzylisoquinolines. Additional structural types are formed through subsequent coupling reactions. For example, the protoberberine backbone is formed by an FAD-dependent oxidoreductase, and the backbone can be further modified by CYPs to form phthalides, benzo[c]phenanthridines, etc. The orange highlighting indicates the coupling atom.

6 1.4 Aldo-Keto Reductases

1.4.1 General Features of AKRs

AKRs form a large protein superfamily of NAD(P)(H)-dependent oxidoreductases. AKRs

predominantly catalyze the reduction of carbonyls and the oxidation of alcohols with notable

exceptions from the AKR1D family that catalyze the irreversible reduction of steroidal C=C

double bonds (Rižner and Penning, 2014; Sengupta et al., 2015). AKRs are commonly 320 amino acids per monomer (34-37kDa), and contain a characteristic (αβ)8-barrel motif with three large loop extensions that are thought to confer substrate specificity (Penning, 2014). Amino acid sequence alignments and structural studies of AKRs have revealed a conserved co-factor binding domain that permits the pro-R-hydride transfer from NAD(P)H to the substrate and vice versa (Penning, 2014).

AKRs are classified according to the AKR nomenclature system that uses amino acid sequence identity for family (>40% sequence identity) and subfamily (>60% sequence identity) classification (Penning, 2014). Additional classification is attained by assigning each subfamily member a number that corresponds to its chronological submission date (Figure 1.3).

Information about AKRs is available online (www.med.upenn.edu/akr).

CYP82Y2 Family, Subfamily, Number AKR4B1

Figure 1.3. CYP and AKR naming convention. The red number indicates the CYP/AKR family number. The green letter denotes the subfamily. The blue number represents the subfamily member.

7 1.4.2 AKRs in Plant Metabolism

AKRs are widely distributed among the prokaryotes and eukaryotes including, animals,

bacteria, fungi and plants. In plants, a limited number of AKRs have been functionally

characterized and are involved in chemical detoxification (Oberschall et al., 2000; Simpson et al., 2009), iron acquisition (Bashir et al., 2006), osmolyte production (Kanayama et al., 1992;

Loescher et al., 1992; Everard et al., 1997; Olsen et al., 2008), embryo development (Bartels et al., 1991), plant microbe interactions (Sallaud et al., 1995; Welle et al., 1991), membrane transport (Tang et al., 1995), and plant specialized metabolism (Unterlinner et al., 1999; Gavidia et al., 2002).

1.4.3 AKRs in BIA Metabolism

In BIA metabolism, four full-length AKR isoforms from the AKR4B family were isolated from opium poppy using a combination of peptide sequencing and PCR (Unterlinner et al.,

1999). Functional characterization of the recombinant AKR proteins revealed oxidoreductase

activity with semi-synthetic and native opium poppy morphinan alkaloids, including the late

morphine pathway intermediates codeinone and morphinone (Unterlinner et al., 1999). In the

context of native opium poppy morphinan substrates, these AKRs preferentially catalyze the

reduction of codeinone and morphinone via a pro-R hydrogen abstraction from NADPH yielding codeine and morphine alcohols, respectively, but also catalyze their oxidation via incorporation of hydrogen into NADP+ yielding codeinone and morphinone carbonyls, respectively. Based on these observations, these AKRs were renamed codeinone reductases 1.1-to-1.4 (COR1.1-1.4), and are responsible for the penultimate and final biosynthetic steps in morphine biosynthesis in opium poppy (Figure 1.4; Unterlinner et al., 1999).

Figure 1.4. Biosynthesis of morphine in opium poppy. Condensation of dopamine and 4HPAA via NCS yields (S)-norcoclaurine. (S)-norcoclaurine undergoes functionalizations to yield the central branch-point intermediate (S)-reticuline. Stereochemical inversion reticuline marks the committed step in morphine biosynthesis, whereby (R)-reticuline undergoes oxidative C-C phenol coupling to form salutaridine. Subsequent modifications to salutaridine yield thebaine. Thebaine can undergo O-demethylation by T6ODM or CODM to yield neopinone or oripavine, respectively. Neopinone spontaneously rearranges to codeinone in aqueous solution and is reduced to codeine by COR. Codeine is demethylated by CODM to produce morphine. Demethylation of oripavine by T6ODM yields morphinone, which is also reduced by COR to morphine. DRS, dehydroreticuline synthase; DRR, dehydroreticuline retductase; SalSyn, Salutaridine synthase; SalR, Salutaridine reductase; SalAt, Salutaridine acetyltransferase; CODM, codeine O-demethylase; T6ODM, Thebaine 6-O-demethylase; COR, codeinone reductase. Dashed lines indicate multiple steps.

9 Interestingly, the RNAi-mediated silencing of the COR gene family in opium poppy led to the unexpected accumulation of the upstream central pathway intermediate reticuline as opposed to COR empirical substrates (Allen et al., 2004). This phenotype was surprising, and was

attributed to a negative feedback response initiated by silencing COR that affected relevant

pathway genes/transport steps or the abolishment of a larger interdependent enzyme complex

through the elimination of COR. An alternative hypothesis that could rationalize this phenotype

is the off-target gene silencing of a COR paralogue. This seems plausible considering that the

biosynthetic route to morphine requires the stereochemical inversion of (S)-reticuline to (R)

reticuline via a proposed NADPH dependent reductive enzyme (Battersby et al., 1965; De-

Eknamkul and Zenk, 1992).

1.5 Cytochromes P450 Monoxygenases

1.5.1 General Features of CYPs

Cytochrome P450 monoxygenases belong to a large protein superfamily with each member

containing the characteristic cysteinato-linked-heme at the centre of a protoporphyrin (IX) ring

(Figure 1.5a). It is the heme-group of each CYP that binds molecular oxygen, which upon activation catalyzes difficult reaction types including, hydroxylations, epoxidations, O- and N

dealkylations, epimerizations, etc (Bernhardt, 2006). As a general scheme, CYPs insert one

atom of molecular oxygen into the substrate, while the other atom is reduced to water (Scheme

1; Meunier et al., 2004). As such, CYPs are termed ‘mono’-xygenases; however, this expression

does not reflect CYP reactions that omit oxygen from the final reaction product. Alternatively,

CYPs have adopted the sobriquet ‘mother nature’s blowtorch’ for their substrate-specific and versatile oxidizing capacity in a variety of biochemical reactions at physiological conditions (Im

and Waskell, 2011).

10 + + SH + O2 + NAD(P)H + H → SOH + H2O + NAD(P)

Scheme. 1

Although there are different methods of delivering electrons from NAD(P)H to the active

CYP core, the majority of CYPs require a reductive protein partner to facilitate the transfer of

electrons from NAD(P)H to CYP for the binding and reduction of molecular oxygen (Figure

1.5b; Meunier et al., 2004). Microsomal CYPs, such as those found in plants, utilize a

membrane bound protein that utilizes a flavin adenine dinucleotide/flavin adenine

mononucleotide electron transfer system for delivering electrons to the CYP.

Like AKRs, CYPs are categorized into families and subfamilies according to a

straightforward nomenclature system based on amino acid sequence identity. As a rule, a CYP

sequence with >40% identity is grouped into the same family whereas a CYP with >55% amino acid identity is placed its corresponding subfamily. To differentiate between each subfamily member, a number is assigned to each protein based on its chronological submission date

(Figure 1.3). Dr. David Nelson is the curator of the worldwide CYP database

(http://drnelson.uthsc.edu/cytochromeP450.html).

CYP reactions are best understood through their multistep catalytic cycle (Figure 1.6). It is generally accepted that the substrate binds to the active site in a location that is nearby the heme-group and opposite to the axial thiolate. This in turn causes a change in the active site conformation that triggers the transfer of one electron from NAD(P)H to the heme-centre.

Electron transfer to the heme centre establishes the ferrous iron state that enables binding of molecular oxygen and the formation of a dioxygen adduct. A second electron is then transferred giving rise to a short-lived peroxo-intermediate that undergoes two successive protonations causing the release of one molecule of oxygen in the form of water and the retention of the other

11 oxygen molecule in the form of a reactive iron-oxo intermediate. The reactive iron-oxo intermediate provides the oxidizing power of CYP enzymes. For the most common CYP

hydroxylation reaction, it is thought that the iron-oxo intermediate forms a complex with the

substrate creating a polar environment that favours the return of water and the preferential

release of the oxidized substrate from the active site (Meunier et al., 2004).

Figure 1.5. Important CYP structures. (A). The heme-group at the centre of the protoporphyrin (IX) ring is essential to the CYP mechanism and is responsible for the binding of molecular oxygen. (B). The majority of CYPs utilize a reductive protein partner for the transfer of electrons from NADPH to CYP. P450R, P450 reductase; FAD, flavin-adenine dinucleotide; FMN, flavin-adenine mononucleotide.

Sequence identity among CYP proteins can be extremely low; however, CYPs share a common overall topology and three-dimensional fold. The highest degree of conservation in

CYPs is found in proximity to the active core, which reflects the common CYP mechanism. The consensus sequence (FxxGxRxCxG) within this region encodes the heme-binding loop located on the proximal face of the heme with the absolutely conserved cysteine amino acid acting as

12 ligand to the heme iron. A second absolutely conserved motif (ExRxR) on the proximal side of

the heme is thought to stabilizes the core structure. The third and final consensus sequence and

one that is considered CYP signature (A/G-GxD/E-T-T/S), corresponds to the proton transfer groove on the distal side of the heme that is involved in oxygen binding and activation (Werck-

Reichhart and Feyereisen, 2000).

1.5.2 CYPs in Plant Metabolism

Since the discovery of the first CYP in the late 1950s, numerous CYPs have been characterized, and are now known from all domains of life and even viruses (Lamb et al., 2009).

In plants, all characterized CYPs to date are bound to the cytoplasmic side of the endoplasmic reticulum via a short hydrophobic N-terminal domain and in some instances a hydrophobic protein loop. However, the signal peptides of annotated CYPs from Arabidopsis suggest alternative anchoring sites for some CYPs including the mitochondria and plastid (Werck-

Reichhart et al., 2002).

In land plants, CYPs can be categorized into four groups based on their biological function including those participating in (a) essential reactions conserved in the plant kingdom (e.g. xanthophyll biosynthesis for light harvesting and photo-protection) (b) core reactions conserved in all land plants (e.g. the biosynthesis of fatty acids and cinnamates involved in physical defense) (c) essential reactions that emerged during angiosperm evolution (e.g. phytohormone metabolism and homeostasis), and (d) specialized reactions that are restricted to one or a small group of plant species (Mizutani and Ohta, 2010).

13 1 2 H2O

- e R-OH R-H

7 3

+ H 6 5 4 H20

+ - H e

Figure 1.6. CYP catalytic cycle. 1. Addition of the prime substrate causes replacement of water leading to 2 where first electron from NADPH is donated giving rise to Fe(II) in 3 that binds with molecular oxygen in 4. The second electron is donated giving rise to a short-lived peroxo intermediate in 5 that undergoes two successive protonations in 6 and 7 that give rise to the reactive iron-oxo intermediate that is responsible for oxidizing the prime substrate. Adapted from: (Guengerich, 2001).

14 1.5.3 CYPs in Secondary Metabolism

In plant specialized metabolism, CYPs are important catalysts that help deliver the vast chemical architectures seen throughout plant specialized metabolism including the anticancer drug taxol (Croteau et al., 2010) and the antimalarial drug artemisinin (Ro et al., 2006) derived from terpenoids, the antihypertensive drug ajmalicine (Collu et al., 2001) from monoterpenoid

indole alkaloids, and other commercially important molecules, including several belonging to the

BIAs (Schäfer and Wink, 2009; Mizutani and Sato, 2011).

1.5.4 CYPs in BIA Metabolism

In BIA metabolism, CYP reactions have so far been associated with three CYP families,

namely CYP80, CYP719 and CYP82 (Mizutani and Sato, 2011). Members of the CYP80 family

catalyze a number of landmark oxidative reactions including C-O and C-C phenol coupling

reactions that give rise to new BIA sub-classes, and hydroxylations that create hydroxy

substituted 1-benzylisoquinoline intermediates that are important for the formation of pathway

end-products. The first member of this family to be identified and characterized was CYP80A1

from Berberis stolinifera (Stadler and Zenk, 1993; Kraus and Kutchan, 1995). CYP80A1 is

berbamunine synthase that catalyzes the regio- and stereo-specific oxidative C-O phenol coupling of two molecules of 1-benzylisoquinoline alkaloids resulting in the formation of (R,S) and (R,R)-dimeric products belonging to the subclass of BIAs known as bis-benzylisoquinolines

(Figure 1.7a). The CYP80A1 reaction is uncommon for CYPs where rather than having the classical monoxygenase activity, CYP80A1 functions as an oxidase whereby oxygen acts as the final electron sink and is reduced to water (Mizutani and Sato, 2011). CYP80G2 from Coptis japonica (Ikezawa et al., 2008) is another member of this family that catalyzes the committed step in aporphine biosynthesis by an oxidative C-C phenol coupling of the 1-bezylisoquinoline

(S)-reticuline giving rise to (S)-corytuberine through diradical coupling (Figure 1.7b). The final

15 characterized member of this family is CYP80B1 from Escholtzia californica (Pauli and

Kutchan, 1998). CYP80B1 acts as a classical CYP ‘mono’xygenase, and catalyzes the 3’ hydroxyation of (S)-N-methylcoclaurine giving rise to 3’-hydroxy-N-methylcoclaurine - the penultimate intermediate to the central BIA branch point intermediate (S)-reticuline (Figure

1.7c).

Figure 1.7. BIA reactions catalyzed by CYP80 family members. (A) CYP80A1 catalyzes the oxidative C-O phenol coupling of (R,S) or (R,R) 1-benzylisoquinoline alkaloids forming bis benzylisoquinolines. (B). CYP80G2 catalyzes the oxidative C-C phenol coupling of (S) reticuline yielding (S)-corytuberine. (C) CYP80B1 catalyzes the hydroxylation of (S)-3’ hydroxy-N-methylcoclaurine forming the central branch-point intermediate in BIA biosynthesis, (S)-reticuline (Adapted from: Mizutani and Sato, 2011).

16 The most common reaction-type by CYPs in BIA metabolism is the oxidative cyclization of an ortho-hydroxymethoxy-substituted aromatic ring, giving rise to a methylenedioxy bridge

(Mizutani and Sato, 2011). To date, only members of the CYP719 family (Figure 1.8a) are capable of this reaction type, and characterized members include canadine synthase (CYP719A1;

Ikezawa et al., 2003), styopine synthase (CYP719A2, CYP719A3; Ikezawa et al., 2003), cheilanthifoline synthase (CYP719A5; Ikezawa et al., 2009), and CYP719A9 (Ikezawa et al.,

2009). The formation of the methylenedioxy bridge is thought to involve hydroxylation of the ortho-methoxy group forming a hemiacetal intermediate that upon loss of water forms a methylene oxonium ion that undergoes cyclization with the lone pair of the adjacent hydroxyl group (Figure 1.8b; Mizutani and Sato, 2011). CYP719B1 is the only other characterized member of this family, and catalyzes the formation of the promorphinan scaffold through the strict regio- and stereo-specific C-C phenol coupling of the 1-benzylisoquinoline (R)-retiucline to form salutaridine (Figure 1.8c; Zenk et al., 1989; Gerardy and Zenk, 1992; Gesell et al., 2009).

This reaction marks the committed step in the synthesis of morphinan alkaloids in opium poppy.

Figure 1.8. CYP719 reactions in BIA metabolism. (A). The most common CYP reaction in BIA metabolism is the formation of the methylenedioxy bridge by CYPs. (B). The proposed formation of the methylene dioxybridge by CYP719s in BIA metabolism. (C). Formation of the promorphinan scaffold by another member of the CYP719 family (Adapted from:Mizutani and Sato, 2011).

The CYP82 family is the only other family to participate in BIA metabolism, and reactions catalyzed by this family have so far been restricted to the incorporation of oxygen into a heterocyclic ring, although oxygen is not always retained in the final reaction product (Mizutani and Sato, 2011). CYP82N4 and CYP82N2v2 catalyze the antepenultimate and penultimate steps, respectively, in the biosynthesis of the antimicrobial BIA sanguinarine (Rueffer and Zenk,

1987; Tanahashi and Zenk, 1990; Takemura et al., 2013; Beaudoin and Facchini, 2013).

CYP82N4 catalyzes the C14 hydroxylation of different N-methylated protoberberines giving rise to their respective protopine alkaloids (Figure 1.9a; (Rueffer and Zenk, 1987; Beaudoin and

Facchini, 2013). CYP82N2v2 catalyzes the ensuing step in the pathway, which is the C6 18 hydroxylation of protopine alkaloids, which triggers a spontaneous rearrangement forming the

benzo[c]phenanthridine alkaloids (Figure 1.9b; Tanahashi and Zenk, 1990; Takemura et al.,

2013). Recently, three other members of the CYP82 family were identified in opium poppy that

contribute to the biosynthesis of the phthalide BIA noscapine (Figure 1.10; Winzer et al., 2012;

Dang et al., 2014). CYP82Y1 catalyzes the initial C1 hydroxylation of N-methylcanadine

forming the requisite backbone for a subsequent C13 hydroxylation by CYP82X2. Following an

acetylation to the C13-hydroxyl moeity, CYP82X1 catalyzes a C8 hydroxylation, which

facilitates a spontaneous ring opening. Three additional enzymatic reactions complete the

biosynthesis of noscapine (Dang et al., 2014).

Figure 1.9. CYP82 family members involved in BIA metabolism. (A). Formation of the protopine and benzo[c]phenanthridine backbones by members of the CYP82 family. (B). CYP82 family members involved in the formation of the antitussive and potential anticancer drug noscapine in BIA metabolism in opium poppy (Adapted from: Takemura et al., 2013 and Dang et al., 2014).

19 1.5.5 The Plant CYP Goldmine

Plant genomes in particular contain a large number of CYP genes. For example,

Arabidopsis thaliana, Oryza sativa and Populus have 246, 356 and 312 predicted CYPs, respectively, accounting for an estimated 1% of their total gene compliment, however, only approximately 25% of Arabidopsis CYPs have been functionally characterized (Nelson and

Werck-Reichhart, 2011). Similarly, considering that thousands of plant specialized metabolites can be rationalized through CYP reactions, the number of unique CYP enzymes awaiting discovery are substantial. In BIA metabolism, there are several BIA sub-classes that can be rationalized through CYP reactions (Figure 1.2), however, only a limited number of CYP enzymes have been functionally characterized.

1.6 2-oxoglutarate/Fe(II)-dependent Dioxygenases

1.6.1 General Features of ODDs

The first reported dioxygenase activity was pyrocatechase from Pseudomanas spp., which catalyzes the 1,2-intradiol cleavage of pyrocatechin to muconic acid (Hayaishi and Hashimoto,

1950). In this reaction, both atoms of molecular oxygen are incorporated into the muconic acid product, leading to use of the term “di”oxygenase (Hayaishi et al., 1956). By definition, dioxygenases have the ability to incorporate two atoms of molecular oxygen into one or more substrates. However, this definition can be ambiguous owing to the number of different dioxygenase types, which vary by the nature of their co-factor and co-substrate dependence. My thesis deals with the mononuclear iron dioxygenases, with particular emphasis on a specific sub class, the ODDs. ODDs are non-heme proteins belonging to a large superfamily that are ubiquitously distributed throughout nature, and occur in bacteria, fungi, plants, and vertebrates

(De Carolis and De Luca, 1994; Martens et al., 2010). The landmark discovery of the first ODD revealed its hydroxylation capacity for prolyl and lysyl amino acid residues, and established the

20 necessary factors required for enzyme function (Hutton et al., 1967). ODDs are dependent on ferrous iron as a co-factor for the binding of molecular oxygen and subsequent oxidative reactions. In essentially all cases, ODDs couple the two-electron oxidation of the prime substrate

[S] to the oxidative decarboxylation of the distinctive co-substrate—2-oxoglutarate (2OG)— giving rise to succinate and carbon dioxide (Scheme 2; Wilmouth et al., 2002; Zhang et al.,

2004; Flashman and Schofield, 2007; Hangasky et al., 2013). The oxidation of the prime [S] substrate leads to the formation of specific products [SO]. In addition to 2OG and ferrous iron,

ODD activity is usually increased by the addition of catalase and ascorbate. Catalase serves as a protecting agent from hydrogen peroxide (Prescott and John, 1996), whereas ascorbate— although not always essential—supports enzyme function by completing spontaneous-uncoupled reactions (Clifton et al., 2006) and is thought to assist with enzymatic cycles by maintaining the ferrous iron state (De Carolis and De Luca, 1994; Prescott and John, 1996). ODDs facilitate numerous oxidative reactions including hydroxylations, halogenations, desaturations, epimerization, cyclizations, and ring formation, ring fragmentation, C-C bond cleavage, rearrangements, demethylations, and demethylenations (Clifton et al., 2006; Flashman and

Schofield, 2007; Loenarz and Schofield, 2008; Tarhonskaya et al., 2014). This impressive list of reactions reveals the versatility of these enzymes in catalyzing many reactions that are still not possible using synthetic chemistry (Flashman and Schofield, 2007). The significance of ODDs is underscored by their widespread roles in biosynthetic pathways essential for normal organismal function, or that lead to high-valued specialized metabolites. The Arabidopsis thaliana genome contains more than 130 ODD genes, representing approximately 0.5% of the total gene complement (Kawai et al., 2014). However, only a handful of plant ODDs have been functionally characterized. Extrapolating from the number of ODD genes in Arabidopsis, a

21 plethora of reactions and roles for ODDs in other plant species can be predicted. Given the known importance of ODDs in plant metabolism, the continued functional characterization of

ODDs is essential.

1.6.2 Structural and Mechanistic Features of 2-oxoglutarate/Fe(II)-dependent Dioxygenases.

Structural analyses of leucoanthocyanidin synthase (LDOX; Wilmouth et al., 2002;

PDB:1GP4), 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO; Zhang et al., 2004;

PDB:1W9Y) and several related ODDs have revealed canonical structural features, including a double stranded β-helix core fold known as jellyroll topology that supports and protects a catalytic triad of Fe(II) binding residues (Clifton et al., 2006). These residues are comprised of a highly conserved, but not ubiquitous, HX(D/E)XnH triad motif that is essential for binding Fe(II)

(Figure 1.10). Residues conferring 2OG binding are less conserved and are usually characteristic of ODD sub-families, whereas those linked to binding of the prime substrate are variable, but might involve features close to the active core (Loenarz and Schofield, 2008). Detailed crystallographic, spectroscopic and kinetic analyses have revealed mechanistic features that are apparently ubiquitous for the formation of the high-valent iron-oxo intermediate responsible for oxidation of the prime substrate. The consensus mechanism involves binding of ferrous iron, which displaces two water molecules and permits the bidentate binding of 2OG (C-1 carboxyl,

C-2 keto). Binding of the prime substrate weakens an additional water molecule and exposes another iron binding site for molecular oxygen. Subsequent decarboxylation of 2OG gives rise to

CO2 and a succinate bound iron-oxo intermediate. The iron-oxo intermediate is essential for oxidation of the prime substrate (Figure 1.11), and has been likened to Fenton chemistry

(Prescott and John, 1996; Groves, 2006). Oxidative mechanisms are specific to each prime substrate, and several different mechanisms have been proposed (Wilmouth et al., 2002). Some

22 atypical ODDs, such as an enzyme involved in ethylene formation, have a 2OG binding motif, but utilize ascorbate instead of 2OG for formation of the reactive iron-oxo intermediate.

1 10 20 30 40 50 60 | | | | | | | PsPODA METAKLMKLGNGMSIPSVQELAKLTLAEIPSRYICTVENLQLP---VGASVIDDHETVPV PsT6ODM MEKAKLMKLGNGMEIPSVQELAKLTLAEIPSRYVCANENLLLP---MGASVINDHETIPV PsCODM METPILIKLGNGLSIPSVQELAKLTLAEIPSRYTCTGESPLNN---IGASVTDD-ETVPV AtLDOX ------MVAVERVESLAKSGIISIPKEYIRPKEELESINDVFLEEKKEDGPQVPT PhACCO ------MENFPI

PsPODA IDIENLISSEPVTEKLELDRLHSACKEWGFFQVVNHGVDTSLVDNVKSDIQGFFNLSMNE PsT6ODM IDIENLLSPEPIIGKLELDRLHFACKEWGFFQVVNHGVDASLVDSVKSEIQGFFNLSMDE PsCODM IDLQNLLSPEPVVGKLELDKLHSACKEWGFFQLVNHGVDALLMDNIKSEIKGFFNLPMNE AtLDOX IDLKNIESDDEKIRENCIEELKKASLDWGVMHLINHGIPADLMERVKKAGEEFFSLSVEE PhACCO ISLDKVNG---VERAATMEMIKDACENWGFFELVNHGIPREVMDTVEKMTKGHYKKCMEQ

PsPODA KIKYG--QKDGDVEGFGQAFVASEDQTLDWADIFMILTLPLHLRKPHLFSKLPLPLRETI PsT6ODM KTKYE--QEDGDVEGFGQGFIESEDQTLDWADIFMMFTLPLHLRKPHLFSKLPVPLRETI PsCODM KTKYG--QQDGDFEGFGQPYIESEDQRLDWTEVFSMLSLPLHLRKPHLFPELPLPFRETL AtLDOX KEKYANDQATGKIQGYGSKLANNASGQLEWEDYFFHLAYPEEKRDLSIWPKTPSDYIEAT PhACCO RFKEL------VASKALEGVQAEVTDMDWESTFFLKHLP--ISNISEVPDLDEEYREVM

PsPODA ESYSSEMKKLSMVLFEKMEKALQVQAVEIKEISEVFKDMTQVMRMNYYPPCPQPELAIGL PsT6ODM ESYSSEMKKLSMVLFNKMEKALQVQAAEIKGMSEVFIDGTQAMRMNYYPPCPQPNLAIGL PsCODM ESYLSKMKKLSTVVFEMLEKSLQL--VEIKGMTDLFEDGLQTMRMNYYPPCPRPELVLGL AtLDOX SEYAKCLRLLATKVFKALSVGLGLEPDRLEKEVGGLEELLLQMKINYYPKCPQPELALGV PhACCO RDFAKRLEKLAEELLDLLCENLGLEKGYLKNAFYGSKGPNFGTKVSNYPPCPKPDLIKGL * * * PsPODA TPHSDFGGLTILLQLNEVEGLQIKNEGRWISVKPLPNAFVVNVGDVLEIMTNGMYRSVDH PsT6ODM TSHSDFGGLTILLQINEVEGLQIKREGTWISVKPLPNAFVVNVGDILEIMTNGIYHSVDH PsCODM TSHSDFSGLTILLQLNEVEGLQIRKEERWISIKPLPDAFIVNVGDILEIMTNGIYRSVEH AtLDOX EAHTDVSALTFILHN-MVPGLQLFYEGKWVTAKCVPDSIVMHIGDTLEILSNGKYKSILH PhACCO RAHTDAGGIILLFQDDKVSGLQLLKDGQWIDVPPMRHSIVVNLGDQLEVITNGKYKSVMH * * PsPODA RAVVNSTKERLSIATFHDP-NLESEIGPISSLITPNTP--ALFRSGSTYGELVEEFHSRK PsT6ODM RAVVNSTNERLSIATFHDP-SLESVIGPISSLITPETP--ALFKSGSTYGDLVEECKTRK PsCODM RAVVNSTKERLSIATFHDS-KLESEIGPISSLVTPETP--ALFKRG-RYEDILKENLSRK AtLDOX RGLVNKEKVRISWAVFCEPPKDKIVLKPLPEMVSVESP--AKFPPR-TFAQHIEHKLFGK PhACCO RVIAQKDGARMSLASFYNP-GSDAVIYPAPALVEKEAEENKQVYPKFVFDDYMKLYAGLK

PsPODA LDGK-SFLDSMRM Region C PsT6ODM LDGK-SFLDSMRI PsCODM LDGK-SFLDYMRM AtLDOX EQEE-LVSEKND------PhACCO FQAKEPRFEAMKAMETDVKMDPIATV Figure 1.10. Sequence alignments of O-demethylases from Papaver somniferum and ODDs from other plants for which crystal structures have been determined. PsCODM, codeine O demethylase; PsT6ODM, thebaine 6-O-demethylase; PsPODA, protopine O-dealkylase; AtLDOX, leucoanthocyanidin dioxygenase; PhACCO, 1-aminocyclopropane-1-carboxylic acid oxidase. Blue asterisks indicate a conserved catalytic triad motif (HXDXnH). Red asterisks indicate proposed 2OG binding residues. Region C contains residues associated with the regiospecific 3-O-demethylation of thebaine (Runguphan et al., 2012).

Figure 1.11. Proposed mechanisms of 2-oxoglutarate binding and formation of the reactive Iron-oxo intermediate for the oxidation of the prime substrate. (I) Ferrous iron binds to the active site of the jellyroll structure. (II) Addition of 2OG displaces two water molecules and permits the bidentate binding of 2OG. Subsequent binding of the prime substrate (SH) in (II) vacates a third binding site for molecular oxygen (III). Oxidative decarboxylation of 2 oxoglutarate (III-IV) yields the reactive iron-oxo intermediate and succinate. (IV-VI) Hydrogen abstraction and two-electron oxidation of the prime substrate leads to the formation of the product (SOH) and the release of succinate for subsequent enzyme cycles (Adapted from: Tarhonskaya et al., 2014).

1.6.3 ODDs in Primary Metabolic Networks

ODDs are involved in several primary metabolic processes including in the repair of deleterious mutations to DNA and RNA (Lindahl et al., 1988; Mielecki et al., 2012), in gene activation or repression (Cho et al., 2012), the post-translational modification of proline residues

(Gorres and Raines, 2010), as oxygen sensors (Vigani et al., 2013) and potentially iron sensors in plants (Jin et al., 2007; Lan et al., 2011), and in the metabolism of several phytohormones including gibberellins (Yamaguchi, 2008), ethylene (Nakajima and Mori, 1990; Van der Straeten et al., 1990), auxin (Zhao et al., 2013) and salicylic acid (Zhang et al., 2013).

1.6.4 ODDs in Plant Specialized Metabolism

In plant specialized metabolism, ODDs are versatile oxidative catalysts that appear throughout numerous plant specialized metabolite pathways. For example, ODDs catalyze key

24 oxidative reactions that facilitate the formation of different flavonoid sub-classes (Martens et al.,

2010), are involved in glucosinolate side chain modification (Halkier and Du, 1997; Kliebenstein et al., 2001; Hansen et al., 2008), catalyze the final two-step conversion of the tropane alkaloid hyoscyamine to the 6,7-epoxide scopolamine (Hashimoto and Yamada, 1986; Hashimoto and

Yamada, 1987), hydroxylate the monoterpenoid desacetoxyvindoline to the immediate precursor of the anticancer drug vindoline (De Carolis et al., 1990; De Carolis and De Luca, 1993;

Vazquez-Flota et al., 1997), are involved in the biosynthesis of the allelophatic benzoxazinoid compound DIBOA (Frey et al., 2003; Jonczyk et al., 2008), are key to the synthesis of coumarin defense compounds (Kai et al., 2008; Matsumoto et al., 2012; Vialart et al., 2012), help synthesize mugineic acid phytosiderophores to chelate iron from the soil (Nakanishi et al., 1993;

Nakanishi et al., 2000; Okumura et al., 1994; Bashir et al., 2006), are involved in the biosynthetic pathway of steroidal glycoalkaloids (Itkin et al., 2013), and were recently found to partake in BIA metabolism (Hagel and Facchini, 2010).

1.6.5 ODDs in Benzylisoquinoline Alkaloid Biosynthesis

CYPs were long considered responsible for the late O-demethylation steps in the branch pathway converting (S)-reticuline to morphine in opium poppy owing to the activity of human

CYP2D6, which catalyzes the reactions (Grobe et al., 2009). Using an opium poppy DNA microarray to compare the transcriptomes of high-morphine and morphine-free opium poppy chemotypes, a gene encoding a ODD was identified from the high-morphine chemotype that was absent from the morphine-free chemotype (Hagel and Facchini, 2010). The recombinant protein catalyzed the 6-O-demethylation of thebaine and oripavine, and the enzyme was named thebaine

6-O-demethylase (T6ODM) (Figure 1.4). Two related ODDs were identified using homology searches of opium poppy transcriptome databases. One, codeine O-demethylase (CODM), showed strict 3-O-demethylation activity with thebaine and codeine (Hagel and Facchini, 2010), 25 while a function was not assigned to the remaining ODD paralogue (DIOX2). Interestingly,

CODM also catalyzed the 6-O-demethylation of the structurally unique protoberberine BIA (S)

scoulerine, which suggested additional roles for CODM, and hinted at a function for DIOX2.

An analysis of BIA scaffolds in the literature would suggest that O-demethylation is widespread throughout BIA metabolism (Hagel and Facchini, 2010b). For example, BIAs that arrive through intermediates with established O-methylation patterns are sometimes missing, suggesting the presence of ODDs in these pathways (Hagel and Facchini, 2010b). In opium poppy, BIA branch pathways outside of morphine contain compounds that are lacking O-methyl groups that were present on established precursors. For example, some protoberberines and protopines derived from (S)-reticuline, which invariably contains a 7-O-/3’-O hydroxyl and 6-O

/4’-O-methoxy pattern; are lacking at least one of these methoxy groups. Similarly, the papaverine analogs pacodine and palaudine are missing the 7-O- and 3’-O-methoxy groups, respectively, that were present on papaverine suggesting O-demethylation as a possible route to these BIAs. O-dealkylation reactions such as O-demethylation are considered rare in plant metabolism; however, in BIA metabolism further investigation is merited based on the presence of metabolites with unexpected O-methoxy substitution patterns.

1.7 Gene Fusions

Genetic recombination events such as translocation, inversion or duplication can create fusion of DNA sequences associated with different genes. These fusion events can lead to new genes from pre-existing parts or, alternatively, shuffle genes into sub-parts across the genome.

Gene fusion events can occur in both the coding and non-coding regions of DNA. The former can lead to mis-regulated gene expression whereby the gene can come under control by the cis- regulatory elements of another gene. Alternatively, gene fusions between the coding regions of two or more independent genes can result in a completely new gene that encodes for a 26 multidomain protein (Durrens et al., 2008). Gene fusions between two independent coding

sequences are often indicative of functional interaction between proteins. For example, fusions

often occur between genes in the same biochemical pathway or protein complex. As such, gene

fusions can be used to predict gene function. For example, if the function of one gene is known,

the function of the neighbouring gene can be predicted based on its association with the known gene (Hanson et al., 2011). Interestingly, protein fusions can provide advantages within a

biochemical pathway. For example, the close proximity of protein domains can lead to the

channeling of pathway intermediates, pathway regulation, and enhanced catalytic efficiency

(Elleuche, 2014). In plants, gene fusions have not been thoroughly investigated; however, the

availability of many sequenced plant genomes and transcriptomes provides a means to identify

novel gene fusions that can help us to understand unknown aspects of plant metabolism.

1.8 Strategies for Gene Discovery in Plant Specialized Metabolism

1.8.1 Traditional Approaches

The pioneering approach for BIA metabolic gene discovery involved native protein

purification, sequencing of the protein, and use of the protein sequence for designing primers that

could be used to amplify and isolate the full-length cognate gene. By obtaining the molecular

clone, protein function could be investigated through heterologous protein expression in a

suitable host. This approach was fundamental for the discovery and characterization of landmark

BIA biosynthetic genes (Dittrich and Kutchan, 1991; Takeshita et al., 1995; Morishige et al.,

2000; Choi et al., 2002; Samanani et al., 2004), whereas adaptations to this approach have led to the discovery of additional BIA genes. For example, primers based on conserved sequence motifs of CYPs were used to isolate the methylene dioxybridge forming enzymes cheilanthifoline synthase and stylopine synthase (Ikezawa et al., 2003; Ikezawa et al., 2007).

Alternatively, heterologous probes designed around known methyltransfereases were used to

27 identify functionally equivalent homologues from opium poppy cDNA libraries (Facchini and

Park, 2003).

1.8.2 Omics Strategies

Traditional approaches have been paramount for the discovery of BIA genes, but suffer

from being tiresome and time consuming. These challenges were largely overcome by

technological advancements in nucleotide sequencing and analytical techniques such as mass

spectrometry and high-performance liquid chromatography. For example, the utilization of

Sanger sequencing for the creation of expressed sequence tag (EST) libraries provided

researchers a means to query gene candidates in silico using programs like BLAST. This homology based approach led to the identification of several BIA genes, and is still used today but with the advantage of larger genome and transcript databases that have been made possible by next generation sequencing technologies such as 454 and illumina. By using a combinatorial approach, gene discovery is improved even further. In this context, BIA genes have been discovered based on their correlation with a certain alkaloid phenotype (Dang et al., 2014). In

addition, technological advancements have enabled the rapid analysis of plant extracts, which

has allowed for the subsequent prediction of biosynthetic pathways. Based on the molecular

structures found in these pathways, reasonable gene candidates can be selected. From here,

candidates can be heterologously produced in a suitable host and screened with appropriate

substrates. In this context, rapid chromatography technologies such as ultra-high pressure liquid

chromatography in tandem with accommodating mass spectrometry platforms have enabled

rapid candidate screening. This is a significant technological advancement, as older techniques

suffer from time consuming methodologies and greater difficulty. My thesis takes advantage of

these techniques to address challenging questions pertaining to BIA biochemistry.

28 1.8.3 Virus-Induced Gene Silencing

Post-transcriptional gene silencing (PTGS) is a viral defense mechanism conserved among

plants and higher eukaryotes (Ruiz et al., 1998; Hannon, 2002). In plants infected with a virus,

the accumulation of aberrant double stranded RNA (dsRNA) during viral replication triggers the

PTGS response that leads to the degradation of viral dsRNA into short interfering RNA (siRNA)

by a highly conserved family of endogenous DICER enzymes. siRNAs are important for

amplifying the PTGS signal but in addition, bind to and activate the RNA-induced silencing

complex that facilitates the degradation of viral RNA sequences and endogenous RNA

transcripts in a homology dependent manner (Hannon, 2002).

PTGS has been exploited to investigate the function of uncharacterized plant genes using a

reverse genetics technique called virus induced gene silencing (VIGS). VIGS relies on the

innate viral response towards aberrant dsRNA. In VIGS, viruses are engineered to carry cDNA

inserts derived from native plant genes. Upon mobilization of the virus into the plant, replication

of the viral cassette triggers the PTGS response that attenuates viral replication and silences the

cognate gene(s) of the cDNA-insert (Ruiz et al., 1998). VIGS is a powerful technique that is

amenable to a variety of recalcitrant plant species for the functional investigation of genes

(Burch-Smith et al., 2004). In this context, several BIA producing plant species including

California poppy (Eschscholzia californica;Wege et al., 2007), columbine (Aquilegia vulgaris;

Gould and Kramer, 2007), meadow rue (Thalictrum flavum; Di Stilio et al., 2010), and opium poppy (Papaver somniferum; Hileman et al., 2005) have utilized VIGS to confirm or establish key steps in BIA biosynthetic networks. In opium poppy, VIGS was first demonstrated using a modified tobacco rattle virus bipartite vector system targeting phytoene desaturase (Hileman et al., 2005). The success of this system for silencing endogenous genes in opium poppy prompted its routine use for investigating the biological roles of BIA biosynthetic genes including those 29 involved in early pathway steps (Lee and Facchini, 2012), morphine biosynthesis (Hagel and

Facchini, 2010a), noscapine biosynthesis (Winzler et al., 2013; Dang et al., 2014), and papaverine biosynthesis (Desgagné-Penix and Facchini, 2012). While alternative transformation techniques have been used to investigate the biological functions of BIA genes in opium poppy

(Allen et al., 2004, Fujii et al., 2007, Frick et al., 2007, Allen et al., 2008, Kempe et al., 2009),

VIGS is comparably easier to perform, requires less time, and is superior for the high-throughput functional analysis of genes (Senthil-Kumar and Mysore, 2014). My thesis utilizes VIGS in concert with advanced analytical techniques to address the objectives outlined below.

1.9 Objectives

The objectives of my thesis were to: i) expand upon earlier investigations of previously reported ODDs from opium poppy ii) to identify and characterize novel ODDs from opium poppy that are involved in BIA metabolism and iii) to identify and characterize the genes responsible for the stereochemical inversion of (S)-reticuline to (R)-reticuline in opium poppy.

The following is summary of how these objectives were obtained:

1. To further investigate 2-oxoglutarate/Fe(II)-dependent dioxygenases from opium

poppy.

New functions were explored for T6ODM, CODM and DIOX2 using VIGS and

biochemical assays with enhanced liquid chromatography tandem mass spectrometry

(LC-MS) detection systems. Observed perturbations to BIAs from VIGS experiments

helped guide in vitro biochemical assays whereby substrates were selected based on these

observations. Additional substrates were tested to define the catalytic range of CODM

and T6ODM. Functions for DIOX2 were determined using available BIA substrates in in

vitro biochemical assays with LC-MS detection. Orbitrap mass spectrometry was utilized

to identify the enzyme products of key biochemical reactions. 30 2. To identify and characterize novel ODDs in opium poppy involved in BIA

metabolism.

Novel ODD candidates were identified using homology-based searches of available

opium poppy transcript assemblies using CODM and T6ODM as queries. New

candidates were cloned and heterologously produced in E.coli and tested for activity with

available BIA substrates. Enzyme activity was monitored using an enhanced LC-MS

detection method and product identities were confirmed using mass spectrometry and/or

nuclear magnetic resonance spectroscopy.

3. To elucidate the genes responsible for the stereochemical inversion reticuline in

opium poppy.

Gene candidates were identified using homology-based searches of available opium

poppy and field poppy transcript assemblies using COR sequences as queries.

Candidates were cloned and heterologously expressed in E.coli or S.cereviceae and tested

for activity with available substrates. VIGS was utilized in opium poppy to investigate

the physiological roles of candidate genes and to corroborate observed in vitro activities.

Chiral chromatography and LC-MS were of vital importance for deciphering the products

of enzymatic reactions.

The experimental findings pertaining to objective i) are presented in Chapter 3. Both Chapter

3 and 4 present findings relating to objective ii). Chapter 5 describes the experimental findings for objective iii). All chapters contain novel data that had not been reported at the onset of my thesis. The enzymes catalyzing the stereochemical inversion of reticuline were reported simultaneously by Winzer et al., 2015 and Farrow et al., 2015.

31 Chapter Two: Materials and Methods

2.1 Plant Material

Seeds of opium poppy (Papaver somniferum L. chemotypes ‘Bea’s Choice’, ‘Veronica’ and ‘T’) and field poppy (Papaver rhoeas) were sown on soil medium consisting of peat, vermiculite and perlite (1:1:1). Plants were cultivated at 20ºC/18ºC (light/dark) with a 16h photoperiod in a growth chamber (Conviron, Winnipeg, Manitoba, Canada; www.conviron.com) under Cool White Fluorescent (Sylvania, Mississauga, Ontario, Canada) and incandescent lights

(Facchini and Park, 2003). The opium poppy variety ‘Bea’s choice’ was cultivated for three weeks in growth chambers before being transferred to a greenhouse for the duration of VIGS experiments. In all cases, plants were grown to maturity with daily watering and weekly fertilization with all-purpose 20-20-20 NPK (Plant-Prod, Brampton, Ontario, Canada).

2.2 Chemicals and Reagents

Restriction endonucleases and Phusion DNA polymerase were from New England Biolabs

(Ipswich, Massachusetts, USA). ExTaq DNA polymerase was from Clontech (Mountain View,

California, USA). Green Taq DNA polymerase was from Genscript (Piscataway, New Jersey,

USA). T4 DNA ligase and Moloney murine leukemia virus reverse transcriptase were from

Invitrogen (Burlington, Ontario, Canada). KAPA SYBR fast was from KAPA Biosystems, Inc.

(Wilmington, Massachusetts, USA). Primers for qRT-PCR and gene isolation were from

Integrated DNA technologies (Coralville, Iowa, USA). Synthetic genes were from GenScript

(Piscataway, New Jersey, USA). d4-methanol was from Cambridge Isotope Laboratories

(Tewksbury, Massachusetts, USA). All other chemicals or reagents were of research or analytical grade and purchased from Sigma (Oakville, Ontario, Canada), VWR (Mississauga,

Ontario, Canada), ThermoFisher (Ottawa, Ontario, Canada), or BioShop (Burlington, Ontario,

Canada).

32 2.3 Benzylisoquinoline Alkaloids

2.3.1 Commercially Available BIAs

Allocryptopine and (S)-scoulerine were purchased from ChromaDex (Irvine, California,

USA). Berberine, papaverine, and sanguinarine were from Sigma. Canadine was from Latoxan

(Valence, France). Codeine and morphine were gifts from Sanofi-Aventis (Paris, France).

Cryptopine was purchased from MP Biomedicals (Santa Ana, California, USA). 1,2 dehydroreticuline iodide, (S)-coclaurine, and (S)-3’-hydroxycoclaurine, were purchased from

Toronto Research Chemicals (Toronto, Ontario, Canada). (S)-Isocorydine, berbamine, (S) boldine, (S)-corytuberine, (S)-glaucine, and (S)-isothebaine were from Sequoia Research

Products (St. James Close, United Kingdom). Protopine was from Idofine Chemicals

(Hillsborough, New Jersey, USA). (R,S)-Tetrahydropalmatine was from Ethnogarden Botanicals

(Barrie, Ontario, Canada). (S)-Reticuline oxalate was a gift from Tasmanian Alkaloids

(Westbury, Australia). (R)-Reticuline was purchased from Santa Cruz biotechnology (Dallas,

Texas, USA). (R,S)-Stylopine (Liscombe and Facchini, 2007) and dihydrosanguinarine (Hagel et al., 2012) were prepared as described previously. Tetrahydropapaverine and pavine were isolated from commercial (±)-pavine (Sigma) as described previously (Desgagné-Penix and

Facchini, 2012).

2.3.2 Opium Derived BIAs

Oripavine and thebaine were isolated as described previously (Hagel and Facchini, 2010) from the latex of opium poppy variety ‘T’.

2.3.3 Enzymatically Prepared BIAs

N-Methyl-1,2-dehydrococlaurine was prepared with ~50% yield from (S)-N methylcoclaurine incubated with NADPH and a microsomal preparation containing Papaver rhoeas dehydroreticuline synthase (PrDRS) and Artemesia annua cytochrome P450 reductase

33 (AaP450R; Ro et al., 2006). (S)-N-methylcoclaurine was prepared with >95% yield from (S)

coclaurine and (S)-adenosyl-L-methionine (SAM) using purified, recombinant Coptis japonica

coclaurine N-methyltranseferase (CjCNMT; Choi et al., 2002). The reaction was basified with ammonium hydroxide and extracted three times with ethyl acetate to remove a bi-product, (S)

N,N-dimethylcoclaurine and cleaned further using solid phase extraction. (S)-3′-Hydroxy-N methylcoclaurine was prepared with ~95% yield from (S)-3′-hydroxycoclaurine and SAM using purified CjCNMT in the presence of sodium ascorbate. Owing to the small quantity of starting material available for biotransformation by PrDRS, 3′-hydroxy-N-methyl-1,2-dehydrococlaurine could not be prepared as a substrate for dehydroreticuline reductase assays. Norcodamine, norlaudanine and norlaudanosine were prepared from (R,S)-norreticuline, and (S)-codamine, (S) laudanine and (S)-laudanosine were prepared from (S)-reticuline, using SAM and O methyltransferases targeting 7-O-methyl and/or 3’-O-methyl groups available in our laboratory

(NCBI Nucleotide accession numbers KP176693 and KP176698). O-desmethylcryptopine was prepared with ~95% yield from cryptopine and 2OG using purified PODA in the presence of

FeSO4 and sodium L-ascorbate. The filtered reaction (0.22μM PVDF filter, Millipore) was

basified with ammonium hydroxide and extracted with ethyl acetate to remove impurities. The

concentration of O-demethylcryptopine in methanol was estimated using the molar extinction

coefficient of cryptopine (Dang et al., 2012). (R,S)-norreticuline was prepared with ~80% yield

from (R,S)-3’-hydroxycocluarine and SAM using purified, recombinant Coptis japonica 3’

hydroxy-N-methylcoclaurine 4’-O-methyltranseferase (Cj4’OMT; Morishige et al., 2000).

(R,S)-3’-hydroxy-N-methylcoclaurine was prepared with ~95% yield from (R,S)-3’ hydroxycoclaurine and SAM using purified CjCNMT in the presence of sodium L-ascorbate.

Pacodine was prepared with ~60% yield from papaverine and 2OG using purified papaverine 7

34 O-demethylase (P7ODM) in the presence of FeSO 4 and sodium L-ascorbate. The filtered

reaction (0.22μM PVDF filter, Millipore) was cleaned using a solid phase extraction cartridge,

and pacodine was isolated using semi-preparative HPLC. α-Hydroxyreticulines were prepared from 1,2-dehydroreticuline iodide using purified and recombinant Papaver somniferum dehydroreticuline reductase 1 (PsDRR1) in the presence of NADPH. The filtered reaction

(0.22µM PVDF filter, Millipore) was cleaned using a solid phase extraction cartridge and α hydroxyreticulines were isolated using semi-preparative HPLC. Identities of prepared alkaloids were confirmed by NMR (Appendix 6 and 7), LC-MS or inferred by comparison with published data (Schmidt et al., 2007; Schmidt et al., 2005; Desgagné-Penix et al., 2012; Farrow et al.,

2012; Wickens et al., 2006). Empirical data and are listed in Appendix 1.

2.4 Solid Phase Extraction

2.4.1 (S)-N-methylcoclaurine

(S)-N-methylcoclaurine was cleaned and concentrated using a Strata-XC cartridge

(200mg.6mL-1, Phenomenex, Torrance, California, USA) equilibrated with methanol and 50mM

phosphate buffer (pH. 7). The sample was resuspended in 50mM phosphate buffer (pH. 7),

loaded onto the cartridge and washed sequentially with water and methanol. (S)-N

methylcoclaurine was eluted in 5% (v/v) ammonia in 70% (v/v) 1:1 methanol:acetonitrile. The

eluent was dried under vacuum and stored at -80°C until later use.

2.4.2 α-Hydroxyreticulines

α-Hydroxyreticulines were cleaned and concentrated using a Strata-X cartridge

(200mg.6mL-1, Phenomenex) equilibrated with methanol and 5% (v/v) ammonia. The sample was basified with ammonium hydroxide (pH. 10), loaded onto the cartridges and sequentially washed with 5% (v/v) ammonium hydroxide and 5% (v/v) methanol. α-Hydroxyreticulines were

35 eluted with (R)-reticuline in 1:1 methanol:acetonitrile. The eluent was dried under vacuum and the residue was stored at -80°C until semi-preparative HPLC.

2.4.3 Pacodine

Pacodine was cleaned and concentrated using a Strata-XC cartridge (200mg.6mL-1 ,

Phenomenex) equilibrated with methanol and 0.1N HCl. The sample was resuspended in water

spiked with 10% 0.1N HCl, loaded onto the cartridge, and washed sequentially with 0.1N HCl

and methanol. Pacodine was eluted with residual papaverine in 5% (v/v) ammonium hydroxide in 1:1 methanol:acetonitrile. The eluent was dried under vacuum and the residue was stored at

80°C until semi-preparative HPLC.

2.5 Semi-Preparative HPLC of Pacodine and α-Hydroxyreticulines

Pacodine and α-hydroxyreticulines were isolated independently using an Agilent 1200

HPLC (Santa Clara, California, USA) equipped with a Luna C18(2) semi-preparative HPLC column (250mm x 10mm; Phenomenex). Sample residue was re-suspended in 0.08% acetic acid containing 5% (v/v) acetonitrile and was injected onto the column in 100µL aliquots. Pacodine and α-hydroxyreticulines were separated with a gradient of 0.08% acetic acid/acetonitrile (95:5, v/v, Solvent A) and acetonitrile (solvent B). The flow rate was set to 5mL.min-1 and initial

conditions were set to 0% solvent B changing linearly to 67% solvent B in 10min. Conditions

were changed immediately at 10.1min to 99% solvent B and remained constant for 1min until

11.2min whereby conditions returned to 0% solvent B. Conditions remained constant for a 5min

re-equilibration period. The elution of pacodine and α-hydroxyreticulines was monitored at

210nm and 284nm. Fractions containing the desired alkaloids were combined in an amber-glass

vial, lyophilized and stored at -80°C until NMR spectroscopy.

36 2.6 Gene Isolation, Expression Vector Construction, Recombinant Gene Expression and Protein Purification

2.6.1 Chapter 3: Identification and Cloning of ODDs

Cloning of CODM (NCBI Nucleotide accession number GQ500141), T6ODM (NCBI

Nucleotide accession number GQ500139), and PODA (NCBI Nucleotide accession number

GQ500140) has been described previously (Hagel and Facchini, 2010). Opium poppy

transcriptome databases (Desgagné-Penix et al., 2012) were searched using the translated

T6ODM (NCBI Nucleotide protein number ADD85329) nucleotide sequence as a query to identify three additional full-length ODD candidates. DIOX4 (NCBI Nucleotide accession number KC854329) and DIOX5 (NCBI Nucleotide accession number KC854330) cDNAs were amplified by RT-PCR from a ‘Bea’s choice’ opium poppy stem cDNA library with Phusion

DNA polymerase and primers with flanking restriction sites (Appendix 2A). Amplicons were

A-tailed using green Taq DNA polymerase. A novel ODD candidate (DIOX6; NCBI Nucleotide accession number KC854331) was amplified from a ‘Bea’s choice’ root cDNA library using

ExTaq DNA polymerase and primers (Appendix 2A) designed for a related sequence found only in an assembled opium poppy root transcript database. PCR products were ligated into pGEM-T easy (Promega, Madison, Wisconsin, USA) and transformed into Escherichia coli XL-1 blue

(Agilent) competent cells. Plasmids were isolated and subsequently digested with restriction endonucleases and fragments were ligated into the pQE30 (Qiagen, Hilden, Germany) expression vector and transformed into E.coli expression strain SG13009 (Qiagen).

2.6.2 Chapter 3: Recombinant Protein Production

Production of recombinant T6ODM, CODM and PODA was performed as described previously (Hagel and Facchini, 2010). Seed culture of E.coli producing DIOX4, DIOX5 and

DIOX6 was grown in LB-broth with selection (≈12-16h) at 37°C with shaking (200rpm), and

37 was used to inoculate up to 1L of LB-broth containing kanamycin and ampicillin. Cultures were

grown to LOG phase whereby protein expression was initiated by the addition of isopropyl β-D

1-thiogalactopyranoside (IPTG) to a final concentration of 0.3mM. Protein expression continued

with shaking (200rpm) at 28°C for 4hrs (DIOX4) or 4°C for 24hrs (DIOX5 and DIOX6).

2.6.3 Chapter 3: Protein Purification

Bacterial pellets were resuspended in protein extraction buffer (100mM TRIS-HCl, pH 7.4,

10% (v/v) glycerol, 14mM-mercaptoethanol (BME)), and cells were lysed at 4°C using a French pressure cell (500psi). After centrifugation to remove insoluble debris, the supernatant containing soluble protein was combined with a buffer-equilibrated Talon resin (Clontech) and shaken on ice (60rpm) for 60min. The protein-charged resin was washed twice with cold (4°C) extraction buffer, and protein was eluted stepwise with increasing concentrations (2.5, 10, 100, and 200mM) of imidazole in extraction buffer. Total proteins (2.5g) from the 100mM imidazole elution were separated by SDS-PAGE to assess protein yield and purity. The 100mM imidazole fraction of each protein was dialyzed overnight using Spectro/Por (Spectrum, Gardena,

California, USA) membranes (15,000 MW cutoff) in 4L of extraction buffer. Proteins were detected by SDS-PAGE (Figure 2.1). Final protein concentrations were estimated using a Bio-

Rad protein assay kit (Bio-Rad, Mississauga, Ontario, Canada), and protein was used immediately or stored at -80°C until later use.

38 42

Figure 2.1. SDS-PAGE of recombinant opium poppy ODD proteins produced in Escherichia coli. Each lane contained approximately 2.5μg of purified, recombinant protein. Visualization was performed by staining with Coomassie Brilliant Blue. Molecular weight protein markers are shown in the left lane.

39 2.6.4 Chapter 4: Identification and Cloning of DIOX7

A novel ODD candidate initially designated DIOX7 was identified by a tBLASTn search

of an opium poppy Bea’s Choice root transcriptome database using the CODM (NCBI Protein

accession number ADD85331) amino acid sequence as a query. DIOX7 (NCBI nucleotide

accession number KT159979) was amplified from Bea’s Choice stem cDNA using Phusion

DNA polymerase and primers listed in Appendix 3A. Amplicons were A-tailed using green Taq

DNA polymerase, ligated into pGEM-T Easy (Promega), and cloned in Escherichia coli XL-1

Blue (Agilent). Isolated plasmids were digested with SacI/KpnI restriction endonucleases and the

DIOX7 fragment was ligated into the pET47b (EMD Millipore, Etobicoke, Ontario, Canada;

http://www.emdmillipore.com/CA/en) expression vector, which was used to transform E. coli

Arctic Express (Agilent).

2.6.5 Chapter 4: Recombinant Protein Production

Seed culture (50mL) of E.coli producing DIOX7 was grown on an orbital shaker (200rpm) at 37°C in LB-broth containing gentamicin, streptomycin and kanamycin, and was used to inoculate up to 1L of LB-broth with the same antibiotic selection. During the logarithmic growth phase, recombinant gene expression was initiated by adding IPTG to a final concentration of

0.3mM. Cultures were grown for 24h at 10°C at 200rpm.

2.6.6 Chapter 4: Protein Purification

Bacterial pellets were re-suspended in protein extraction buffer (100mM Tris–HCl, pH 7.4,

10% (v/v) glycerol,10mM BME, 1mM phenylmethanesulfonyl fluoride (PMSF)), and cells were lysed at 4°C using a French pressure cell (1000psi). Recombinant protein was purified as described previously (Farrow and Facchini, 2013) and desalted using a PD-10 column (GE

Healthcare Life Sciences) with elution in 100 mM Tris–HCl, pH 7.4, 10 mM BME, and 10%

(v/v) glycerol. Proteins were visualized by SDS–PAGE and recombinant DIOX7 was detected by

40 immunoblot analysis as described previously (Dang et al., 2014) (Figure 2.2). Protein concentration was determined using a BCA protein assay kit (Thermo), and purified proteins were used immediately or stored at -80°C for later use.

Figure 2.2. SDS-PAGE and immunoblot analysis of purified recombinant P7ODM produced in Escherichia coli

2.6.7 Chapter 5: Identification and Cloning of AKRs and CYPs

The codeinone reductase gene (COR1.1; NCBI Nucleotide accession number AF108432) was used to query Papaver somniferum stem transcriptome databases by tBLASTn, and two

AKR candidate contigs (AKR1 and AKR2) were identified showing considerable sequence identity to COR1.1. The AKR1 contig displayed an extension that annotated as a CYP, upstream and in-frame with the AKR coding region. Transcriptome databases in the PhytoMetaSyn

(http://www.phytometasyn.com) and the 1000 Plants41

41 (https://www.bioinfodata.org/Blast4OneKP/home) projects were queried using the predicted

CYP-AKR translation product, and full-length orthologs were identified in Papaver setigerum and Papaver bracteatum. In contrast, independent orthologs of the CYP and AKR domains were found in Papaver rhoeas with no evidence of fusion.

Coding regions for the Papaver somniferum AKR1 domain (renamed PsDRR1) and

PsAKR2 and for the Papaver rhoeas AKR (renamed PrDRR) were amplified from corresponding stem cDNA libraries using Phusion DNA polymerase and primers listed in

Appendix 4A. Amplicons were A-tailed using green Taq DNA polymerase and cloned into pET47b. Constructs were used to transform the E. coli expression strains Arctic Express

(PsDRR1 and PsAKR2) or Rosetta (PrDRR; Novagen). The coding region for Papaver somniferum CYP-AKR1 (renamed PsREPI) and the isolated CYP domain (renamed PsDRS) and for the Papaver rhoeas CYP (PrDRS) were synthesized for insertion into the NotI and SpeI restriction sites of the dual expression vector pESC-leu2d (Agilent) along with Artemisia annua cytochrome P450 reductase (CPR; NCBI Nucleotide accession number DQ318192). The expression constructs were used to transform the protease-deficient Sacchoromyces cerevisiae

YPL154c strain (PEP4).

2.6.8 Chapter 5: Recombinant Protein Production in Sacchoromyces cerevisiae

Yeast cultures were transformed using the lithium acetate method, and freshly transformed yeast cultures harbouring pESC-leu2d constructs were grown on an orbital shaker (250rpm) to

LOG phase (≈16h) in 500mL of Synthetic Complete dropout medium supplemented with 2%

(w/v) glucose and lacking leucine (SC-leu). Cultures were centrifuged (4,000g) and resuspended in 1L of protein induction medium containing SC-leu supplemented with 0.2% (w/v) glucose, 1%

(w/v) raffinose and 1.8% (w/v) galactose. Cultures were grown for an additional 6h at 28°C and cells were harvested by centrifugation (4000g). Microsomes were prepared based on a standard 42 protocol (Pompon et al., 1996). Briefly, after initial treatment with TEK buffer (5mM TRIS-

HCl, pH 7.5, 0.5mM EDTA, 100mM NaCl, 20mM KCl, 10mM MgCl2), cells were re-suspended in 5mL of cold TESB (50mM TRIS-HCl, pH 7.5, 150mM NaCl) in a 50mL tube. Ice-cold

0.5mm glass beads (ThermoScientific) were added up to the meniscus and cells were lysed using

1min rounds of vigorous shaking with intermittent incubations on ice for 1min. The process was performed for 4-7 rounds or until at least 70% of the cells were lysed. Glass beads were rinsed

4x with 5mL of TESB, insoluble debris was removed by centrifugation at 15,000g and the supernatant was subjected to sucrose-gradient separation of microsomes by ultracentrifugation for 1h at 45,000g and 4°C. Microsomal pellets were re-suspended in 1mL of 50mM HEPES, pH. 7.5, and recombinant proteins were detected by immunoblot analysis (Figure 2.3A; Dang and Facchini, 2014) and assayed immediately.

2.6.9 Chapter 5: Recombinant Protein Production in Escherichia coli

Cultures of E. coli harbouring pET47b were grown on an orbital shaker (200rpm) to logarithmic growth phase in 1L of LB-broth containing gentamicin, streptomycin and kanamycin

(Arctic Express) or chloramphenicol and kanamycin (Rosetta). Recombinant His-tagged protein production was initiated by the addition of 1mM IPTG. To produce PsDRR1 and PsAKR2, cultures were grown for 24h at 15°C, whereas cultures were grown for 4h at 28°C to produce

PrDRR. For purification, bacterial pellets were resuspended in protein extraction buffer (100mM sodium phosphate buffer, pH 7.4, 10% (v/v) glycerol, 300mM NaCl, 1mM PMSF), and cells were lysed at 4°C using a French pressure cell (1000psi). After centrifugation (10000g) to remove insoluble debris, the supernatant was incubated on ice with buffer-equilibrated Talon resin on an orbital shaker (60rpm) for 60min. The protein-charged resin was washed twice with cold extraction buffer containing 2.5mM imidazole, and proteins were eluted stepwise with increasing concentrations (10 to 200mM) of imidazole in extraction buffer. Total proteins from a 43 40mM imidazole elution were desalted using a PD-10 column and 100mM sodium phosphate

buffer (pH 7.4, 10% (v/v) glycerol). Recombinant proteins were analyzed by SDS-PAGE

(Figure 2.3B) to assess yield and purity, and immunoblot analysis was performed using α-His

primary antibodies (Genscript, catalog number A00186-100; 1:10,000 dilution) and goat

antimouse, horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, catalog number

170-5047; 1:10,000 dilution; Figure 2.3C). Total protein concentration was determined using a

BCA Protein Assay kit and calculated protein concentrations were adjusted based on gel densitometry. Purified proteins were used immediately or stored at -80°C for later use.

Figure 2.3. Heterologous production and/or purification of recombinant enzymes. (A) Immunoblot analysis showing α-FLAG-tagged PsREPI, PsDRS and PrDRS, and α-c-Myc-tagged cytochrome P450 reductase (CPR) in microsomal fractions of Saccharomyces cerevisiae. Recombinant PsREPI, PsDRS and PrDRS were detected using α-FLAG antibodies, whereas recombinant CPR was detected using α-c-Myc antibodies. Each lane contained 2µg of total microsomal protein. (B) Coomassie Blue-stained, denaturing polyacrylamide gel showing α-His tagged PsDRR1, PrDRR and PsAKR2 (arrowhead) produced in Escherichia coli and purified using cobalt-affinity chromatography. (C) Immunoblot analysis showing recombinant, purified PsDRR1, PrDRR and PsAKR2 detected using α-His antibodies.

2.7 Enzyme Assays

2.7.1 Chapter 3

Twenty-seven BIAs were screened in triplicate as potential substrates for opium poppy 2

ODDs using a standardized assay. For each screen, 100ng.µL-1 of purified and desalted protein was incubated for 16h at 30°C with 100µM of candidate substrate, 500µM 2OG, 500µM iron sulfate, and 10mM sodium L-ascorbate in 100mM TRIS-HCl, pH 7.4, containing 10% (v/v) glycerol, 14mM BME, and 5% (v/v) ethanol in a total reaction volume of 100µL. Denatured proteins were used as negative controls. Assays were quenched with 5 volumes of 1M acetic acid in methanol. Samples were reduced to dryness in a speed-vacuum concentrator and resuspended in 1.5ml of HPLC running buffer (10mM ammonium acetate (pH 5.5)/ acetonitrile,

95:5 (v/v)). BIAs accepted as substrates were used to determine relative conversion rates using identical assay conditions except for an incubation time of 45min, which was within the linear range of each assay, a final alkaloid substrate concentration of 50µM, and a total reaction volume of 50µL. Samples were diluted to 1.25ml with HPLC running buffer. (S)-Scoulerine and cryptopine were used as substrates to determine kinetic parameters for CODM and PODA, respectively, at concentrations up to 500µM. Samples were diluted with running buffer to a final substrate concentration of 1µM for analysis by LC-MS.

2.7.2 Chapter 4

Standard DIOX7 assays were performed as described previously for other ODDs with denatured proteins serving as negative controls (Farrow and Facchini, 2013). Thirty-two BIAs were screened in triplicate as potential substrates for DIOX7. Optimal pH and temperature were determined using otherwise standard assay conditions with papaverine as the BIA substrate.

Assays were quenched with six volumes of acetonitrile, reduced to dryness in a vacuum concentrator and stored at -20°C prior to analysis. Kinetic parameters for DIOX7 were

determined using papaverine at concentrations up to 400μM and a fixed concentration of 500μM

2OG. Briefly, assays were incubated at 25°C in 100mM glycine buffer (pH 8.0) containing 100

-1 ng.μL recombinant DIOX7 protein, 10mM sodium ascorbate, 500μM FeSO4, and10mM BME.

Assays were quenched with six volumes of acetonitrile, evaporated in a vacuum concentrator and stored at -20°C until analysis.

2.7.3 Chapter 5: Reductase Assays

Standard reduction (forward) reaction assays included 50ng.µL-1 purified recombinant

protein, 20µM alkaloid and 500µM NADPH in 100mM sodium phosphate buffer, pH 7, in a total

volume of 50µL. Incubation times were 45s (PsDRR1) or 4min (PrDRR) at 37°C. For oxidative

(reverse) reaction assays, 20µM alkaloid and 500µM NADP+ were used as substrates in 100mM

citrate-NaOH buffer, pH 8.8. Denatured recombinant proteins served as negative controls.

Reactions were quenched with 6 volumes of acetonitrile and reduced to dryness. For forward

reactions, kinetic parameters were determined using 1,2-dehydroreticuline iodide at

concentrations up to 300µM and at a fixed concentration of 500µM NADPH. For reverse

reactions, kinetic parameters were measured using (R)-reticuline at concentrations up to 300µM and at a fixed concentration of 500µM NADP+. Substrate range experiments were conducted using the same assay conditions.

2.7.4 Chapter 5: Cytochrome P450 Assays

Standard CYP assays were performed using 25µL of prepared microsomes, 2µM alkaloid and 250µM NADPH in 50mM HEPES, pH 7.5, in a total volume of 50µL. Incubations were for

1h at 37°C. Microsomes prepared from Saccharomyces cerevisiae YPL154c harbouring pESC

leu2d containing only the Artemisia annua CPR served as the negative control. Assays were

quenched with 6 volumes of acetonitrile and reduced to dryness. Kinetic parameters were

determined using up to 64µM (S)-reticuline and at a fixed concentration of 250µM NADPH.

Substrate range experiments with different BIA substrates were conducted using the same assay

conditions.

2.8 Analysis of Enzyme Assays

2.8.1 Chapter 3

Enzyme assays were analyzed by LC-MS using a 1200 HPLC (Agilent) coupled to a 6410 triple-quadrupole MS (Agilent). For substrate range and relative activity analyses, 1 and 10µL of sample, respectively, was injected onto a Zorbax SB C18 HPLC column (1.8µm, 2.1 mm x 50 mm; Agilent), and analytes were eluted with a gradient of running buffer (10mM ammonium acetate (pH 5.5):acetonitrile, 95:5 (v/v) (solvent A), and acetonitrile (solvent B). The flow rate was 700µL.min-1, and the gradient began with 0% solvent B and increased linearly to 99%

solvent B by 7min. The mobile phase composition remained constant until 8.1min at which time

it returned immediately to 0% solvent B followed by a 2min re-equilibration period. Analytes

were subjected to positive electrospray ionization (ESI+) using source conditions optimized for

BIAs (gas temperature, 350 °C; gas flow rate, 10L.min-1; nebulizer gas pressure, 50psi; capillary

voltage, 4000V; fragmentor voltage, 110V; Farrow et al., 2012) and were subsequently detected by full scan MS operating in positive polarity. Quadrupole 1 and 2 were set to RF only with quadrupole 3 scanning from 200-700 mass-to-charge (m/z). Relative enzyme activity was

calculated as the percent turnover of each substrate using the following formula: product peak

area /(substrate peak area + product peak area) x 100. Subsequently, the compound with the

highest turnover for each ODD was set to 100%, and the detected activity with all other

substrates was expressed as a percentage of the maximum. For kinetic parameter analyses, a

five-point calibration curve of cryptopine and (S)-scoulerine (500pM to 5μM) was established to

semi-quantitatively determine the amount of product formed per unit of time. Kinetic constants

were calculated based on Michaelis-Menten kinetics using GraphPad Prism 5 (GraphPad

Software, La Jolla, California, USA).

2.8.2 Chapter 4

Enzymes assays were analyzed by LC–MS as described previously (Farrow and Facchini,

2013). For kinetic parameter analyses, a five-point calibration curve for papaverine (500pM to

5µM) was used to determine the amount of product formed per unit of time. Kinetic constants were calculated based on Michaelis–Menten kinetics using Microsoft Excel and GraphPad Prism

2.8.3 Chapter 5

Enzyme assays were analyzed using an Agilent 1200 HPLC coupled to an Agilent 6410B triple-quadrupole MS. Samples were re-suspended to 1µM in solvent A (0.08% (v/v) acetic acid:acetonitrile (95:5)), and 2µL was injected onto a Hypersil gold SB C18 column (1.8µm, 2.1 mm x 50 mm, ThermoScientific). Analytes were eluted using a gradient of solvent A and solvent B (100% acetonitrile) and at a flow rate of 600 µL.min-1 . Gradient conditions were as

follows: solvent B ramped linearly from 0 to 50% (v/v) over 6min; solvent B increased linearly

to 99% by 7min and remained constant until 8.1min solvent B then returned immediately to 0%

followed by a 3min re-equilibration period. Analytes were subjected to positive ion electrospray

ionization using optimized source conditions (gas temperature, 350°C; gas flow rate, 10L.min-1;

nebulizer gas pressure, 50psi; capillary voltage, 4000V; fragmentor voltage, 110V) and were

subsequently detected by full scan MS operating in positive polarity. Quadrupole 1 and 2 were

set to RF-only with quadrupole 3 scanning from 200-700 m/z. Relative enzyme activity was

calculated as the percent turnover of each substrate using the formula: product peak area /

(substrate peak area + product peak area) x 100. Subsequently, the compound with the highest

turnover for each protein was set to 100% and the activity of other substrates was expressed as a

percentage of the maximum. For kinetic analyses, a five-point calibration curve of 1,2

dehydroreticuline or (R)-reticuline (500pM to 5µM) was established. For chiral separations,

assays were analyzed by HPLC-UV using an Agilent 1200 HPLC coupled to a diode array

detector. Samples were re-suspended to 10µM in 20mM ammonium bicarbonate including 0.1%

(v/v) diethylamine (solvent A), and 20µL was injected onto a Cellulose-1 chiral column (2.1mm

x 150mm, Phenomenex). Analytes were eluted for 30min at a flow rate of 250µL.min-1 using isocratic conditions (80% (v/v) solvent A and 20% acetonitrile (solvent B)) and monitored at

284nm. For kinetic analyses, a five-point calibration curve of (R)-reticuline (10nM to 100µM)

was established. Constants were calculated based on Michaelis-Menten kinetics using GraphPad

Prism 5 software.

2.9 Reaction Product Identification

2.9.1 Triple Quadrupole Mass Spectrometry Analysis

Comparison of collision induced dissociation (CID) spectra with empirical spectra either

from authentic standards or available in the literature (Schmidt et al., 2005; Wickens et al., 2006;

Schmidt et al., 2007; Farrow et al., 2012; Desgagné-Penix et al., 2012) was used to identify or

characterize each enzymatic reaction product (Appendix 1 and 5A). CID was performed at

25eV for each compound, and fragment ions were detected between 40 m/z and 5 atomic mass

units above the m/z of the quasi-molecular ion. In some cases, unequivocal product

identification was not possible because of the occurrence of identical mass spectra for

compounds with O-linked methyl groups occupying one of two positions on either the A-ring of

the isoquinoline moiety or the C-ring of the benzyl moiety. Furthermore, reaction product

annotation was inferred when yields were low and neither an authentic standard nor published

spectra was available. As an example of the inference process, the reduced m/z of (S)-N

methylcoclaurine by 2 atomic mass units in assays with PsDRS or PrDRS implied a two-electron

oxidation yielding N-methyl-1,2-deydrococlaurine. The empirical CID spectra of the inferred N

methyl-1,2-deydrococlaurine reaction product also displayed similar diagnostic features

compared with the 1,2-dehydroreticuline authentic standard. In chapter 2 and 3, the reaction

products of enzyme assays with papaverine and P7ODM or PsDRR1 and α-hydroxy-1,2

dehydroreticulines were confirmed using nuclear magnetic resonance spectroscopy (NMR).

2.9.2 LTQ-Orbitrap XL Analysis

High resolution MS and MS2 analysis was performed and was used as an additional

diagnostic tool for the identification of reaction products in enzyme assays with ODDs from

chapter 3 and allocryptopine. Analysis was performed using an LTQ-Orbitrap XL

(ThermoFisher) equipped with a syringe pump. Allocryptopine and enzymatic reaction products

(1mg.mL-1 in acetonitrile, 0.1% (v/v) acetic acid, 50:50) were introduced continuously with a

syringe pump (5µL.min-1) into the electrospray ionization (ESI+) source, and positive ions were

generated using the following parameters: sheath gas, 10 arbitrary units; auxiliary gas, 0 arbitrary

units; sweep gas, 0; spray voltage, 4500V. Ion interface settings were 275°C and 40V (capillary)

and 255V (tube lens). MS2 experiments were performed by conducting CID on target ions

isolated in the linear ion trap followed by high-resolution (60,000 full-width half-mass) mass

analysis of the resulting product ions in the Orbitrap. High-resolution data were collected in

centroid mode over mass ranges bracketing the precursor ion (± 20 atomic mass units of the

predicted m/z) or product ions (from 165 to 220 m/z). External and internal instrument calibrations ensured an error of 2 ppm.

2.9.3 NMR of α-hydroxyreticulines

The dried solid was dissolved in 220μL d4-methanol and placed in an 8” 3mm tube for

NMR analysis. NMR spectra were acquired immediately at 25°C using an Agilent DD2

spectrometer (υ(1H) = 699.758 MHz, υ(13C) = 175.973 MHz) equipped with a 5mm variable

temperature 1H-19F{13C/15N} Triple Resonance Cold Probe and VnmrJ4.0 acquisition software.

1D 1H spectra were acquired using the standard VnmrJ4.0 s2pul pulse sequence. The spectrum was acquired over a 7142.9 Hz spectral window with 71154 points, a 5s recycle delay, and 32 transients. 2D 1H/13C HSQC spectra were acquired at 25ºC using the default VnmrJ4.0 gc2hsqcse pulse sequence. The spectra were acquired with a 7142.9 Hz spectral window and

2048 points in the direct dimension and a 35195.8 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64s recycle delay, and 4 transients.

2D 1H/13C HMBC spectra were acquired using the default VnmrJ4.0 gHMBCAD pulse sequence. The spectra were acquired over a 5733.9 Hz spectral window and 2048 points in the direct dimension and a 42238.6 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64s recycle delay, and 32 transients. 2D COSY spectra were acquired using the default VnmrJ4.0 gCOSY pulse sequence. The spectra were acquired over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 7142.9

Hz spectral window and 512 increments in the indirect dimension. Spectra were collected with a

0.64s recycle delay, and 2 transients. 2D ROESY spectra were acquired using the default

VnmrJ4.0 ROESY pulse sequence. The spectra were acquired over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 7142.9 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 1.5s recycle delay, and 16 transients.

2.9.4 NMR of Pacodine

The dried solid was dissolved in 220μL d4-methanol and placed in an 8”x 3mm NMR tube.

NMR spectra were acquired immediately at 25°C using an Agilent DD2 spectrometer (υ(1H) =

699.809 MHz, υ(13C) = 175.983 MHz) equipped with a 5mm variable temperature 1H

19F{13C/15N} Triple Resonance Cold Probe and VnmrJ4.0 acquisition software using the standard sequences. The 1D1H spectrum was acquired over a 7142.9 Hz spectral window with 52

71154 points, a 5s recycle delay, and 32 transients. The 1D13C spectrum was acquired over a

41666.7 Hz spectral window with 250000 points, a 0.2s recycle delay, and 512 transients.

2D, 1H/13C HSQC spectra were acquired at 25ºC using the default VnmrJ4.0 gc2hsqcse pulse sequence. The spectra were acquired with a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 35195.8 Hz spectral window and 128 increments in the indirect dimension. Spectra were collected with a 0.64s recycle delay, and 4 transients. 2D1H/13C

HMBC spectra were acquired using the default VnmrJ4.0 gHMBCAD pulse sequence. The spectra were acquired over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 42238.6 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64s recycle delay, and 8 transients. 2D COSY spectra were acquired using the default VnmrJ4.0 gCOSY pulse sequence. The spectra were acquired over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 7142.9 Hz spectral window and 256 increments in the indirect dimension. Spectra were collected with a 0.64s recycle delay, and 2 transients. 2D ROESY spectra were acquired using the default VnmrJ4.0 ROESY pulse sequence. The spectra were acquired over a 7142.9 Hz spectral window and 2048 points in the direct dimension and a 7142.9 Hz spectral window and 256 increments in the indirect dimension.

Spectra were collected with a 1.5s recycle delay, 8 transients, and a 0.2s mixing time.

2.10 NMR Processing

NMR spectra were processed using MestReNova NMR processing software (v10.0.1). All spectra were Fourier transformed, phase corrected, and baseline corrected. Window functions were applied as necessary prior to Fourier transformation and all 2D spectra were two-fold linear predicted in the F1 dimension prior to Fourier transformation. Chemical shifts were referenced

1 13 relative to the residual solvent peaks (d4-methanol, δ( H)=3.31 ppm, δ( C)=49.00 ppm.

Appendix 6 and 7 contain a summary of the NMR data. 53

2.11 Reaction Mechanism Assays

2.11.1 Nash Assay

Production of formaldehyde as a by-product of ODD-catalyzed O-demethylation was monitored using a fluorescence-based Nash assay (Hagel and Facchini, 2010a). Nash reagent was prepared by adding 0.3mL of acetic acid and 0.2mL of acetylacetone to 100mL of 2M ammonium acetate. Enzyme assays were performed at 30°C for 45min using 500µM of BIA substrate, 500µM 2OG, 500µM FeSO4, 10mM sodium L-ascorbate, and various quantities of up to 200µg of CODM and PODA in 100mM TRIS-HCl, pH 7.4, containing 10% (v/v) glycerol,

14mM BME, and 5% ethanol (v/v) in a total reaction volume of 500µL. Assays were quenched with 2 volumes of Nash reagent followed by a 10min incubation at 60°C to convert formaldehyde to diacetyldihydrolutidine, which was detected by fluorescence using a

CaryEclipse fluorescence spectrophotometer (Varian, Palo Alto, California, USA) at λ ex 412nm and λem 505nm.

2.11.2 Formate Dehydrogenase Assay

Cryptopine (500µM) was incubated at 30°C for 16h with 200µg of CODM with 500µM

2OG, 500µM FeSO4, and 10mM sodium L-ascorbate in 100mM TRIS-HCl, pH 7.4, containing

10% (v/v) glycerol, 14mM BME, and 5% ethanol (v/v) in a total reaction volume of 500µL. The release of formic acid was monitored by adding 1 unit of formate dehydrogenase and 10mM

NAD+ to the enzyme assays after 16h of incubation. In the presence of formate dehydrogenase

+ and NAD , formic acid undergoes stoichiometric conversion to CO2 and NADH, the latter of which was monitored at 30°C using a Cary Eclipse UV spectrophotometer (Varian) at λ 340nm.

2.12 Virus-Induced Gene Silencing

2.12.1 Vector Construction and Methodology

Virus-induced gene silencing was performed using the tobacco rattle virus (TRV) vector

system (Dinesh-Kumar et al., 2003). Construction of vectors to perform VIGS targeting

T6ODM (pTRV2-T6ODM), CODM (pTRV2-CODM), and both genes (pTRV2-DIOX) has been

described previously (Hagel and Facchini, 2010) and the primers used for the construction of

VIGS constructs are listed in Appendix 2B. In chapter 5, unique or conserved sequences

encompassing parts of the 5’-untranslated region (pTRV2-PsREPI-5’) or open reading frame

(pTRV2-COR1.1 or pTRV2-a) were used for the construction of pTRV2 vectors to suppress the

COR gene family or PsREPI. Fragments of COR1.1 and PsREPI were amplified using primers

with flanking restriction sites listed in Appendix 4B. Amplicons were individually cloned into

pGEM-T-easy and sub-cloned into pTRV2. Assembled pTRV2 constructs were individually

transformed into Agrobacterium tumefaciens strain GV3101 using electroporation, screened on

plates containing kanamycin, and transformation was confirmed by sequencing. Seed cultures

from single colonies grown in kanamycin were used to inoculate up to 500mL of LB

supplemented with 10mM 2-(-N-morpholino) ethanesulfonic acid (MES), 20μM acetosyringone,

and 50μg.mL-1 kanamycin. Cultures were incubated overnight at room temperature with shaking

(200rpm). Bacteria were collected (3000g, 15min) and the pellet was re-suspended in infiltration

solution (10mM MES, 200μM acetosyringone, 10mM MgCl2) to an A600 of 2.5. Each A.

tumefaciens harbouring an assembled pTRV2 construct was mixed 1:1 (v/v) with A. tumefaciens harbouring pTRV1 and left for 3h at room temperature before infiltration. Apical meristems and young leaves of 2-to-3-week-old seedlings were infiltrated using a syringe. Empty pTRV2 was used as a negative control, and the pTRV2-PDS construct encoding phytoene desaturase was used as a positive infiltration control. Infiltrated plants were cultivated in the greenhouse for 8

to-12 weeks. Latex, root and stem samples were harvested for alkaloid and transcript analyses.

Typically, 20 to 30 plants were infiltrated with A. tumefaciens harbouring pTRV1 and one pTRV2 construct. In approximately 70 to 80% of the infiltrated plants, a mobilized fragment of the pTRV2 construct was detected by RT- PCR using the primers listed in Appendix 8. VIGS experiments were performed in duplicate with essentially identical results.

2.12.2 Real Time Quantitative PCR

Experimental plants infected with a pTRV2 construct targeting a BIA gene or infected with the empty pTRV2 construct were subjected to real time quantitative PCR (qRT-PCR) to determine the abundance of relevant mRNAs. qRT-PCR was performed using a 7300 Real-Time

PCR system (Applied Biosystems, Foster City, California, USA; www.appliedbiosystems.com) and the SYBR Green detection method. Each 10μL reaction contained 200nm of each gene specific forward and reverse primers (Appendix 2C; Appendix 3B; Appendix 4C), 2.5μL of

KAPA SYBR mix, and 1μl of cDNA template derived from the stem of the infected plant. The qRT-PCR method consisted of a 3min activation period at 95ºC followed by 40 cycles of denaturation at 95ºC for 3s and annealing/extension/data acquisition at 60ºC for 30s.

Dissociation analysis of the qRT-PCR products was carried out to validate the specificity of each primer pair, and was achieved using the manufacturers protocol (Applied Biosystems).

Reference genes for qRT-PCR were actin (Chapter 3), and actin, ELF-1a and UBQ10 (Chapter

5). Results for reference genes in Chapter 5 yielded essentially identical results for all target genes. Results of Chapter 3 are presented using actin as the reference gene. Results of Chapter 5 are presented using ELF-1a as the reference gene. Efficiencies for all primer sets were approximately equal and always >90%. Relative transcript levels were determined using the 2

ΔΔCt method (Livak and Schmittgen, 2001) and statistical differences between sample groups

were determined using a non-paired, two-way t-test. 56

2.12.3 Alkaloid Extraction

Opium poppy latex was collected in a pre-weighed 2mL tube, centrifuged at 14,000g for

1min at 4°C, and immediately flash-frozen in liquid nitrogen. Samples were lyophilized for 2

days, and the dry weight of the latex was determined. Alkaloids were extracted in cold methanol

(30mL.mg-1 dry weight) and subjected to vigorous vortex mixing and sonication for 1min.

Extracts were incubated for 16h at -20°C and subsequently centrifuged at 14,000g for 10min at

4°C to remove debris. The supernatant was transferred to a new tube and 1:1,000 and 1:10,000

dilutions were prepared in 2ml glass auto-sampler vials using HPLC running buffer for LC-MS

analysis. Opium poppy roots were flash-frozen in liquid nitrogen, lyophilized, and ground to a

fine powder with a TissueLyser II (Qiagen) at 30Hz for 1min using a 30mL grinding jar

precooled in liquid nitrogen and a 10mm stainless steel grinding ball. Approximately 200mg of

powdered root was extracted using the protocol described for latex, and a 1:50 dilution was

prepared for LC-MS analysis.

2.12.4 VIGS Analysis

Samples (10μl) were injected onto a Zorbax SB C18 HPLC column (1.8μm, 2.1mm x

50mm; Agilent), and compounds were separated using the HPLC mobile phase solutions listed

above with a modified gradient. The flow rate was 500μL.min-1, and the gradient began with 0%

solvent B, which was increased linearly to 7.5% by 1.5min and was changed immediately to 25%

at 1.6min. Solvent B was increased linearly to 33% by 5min, 92% by 7min, and 98% by 7.6min.

Solvent B remained constant at 98% until 8.6min and was changed immediately to initial

conditions at 8.7min for a 4min re-equilibration period. Analytes were detected by LC-MS as described for the analysis of enzyme assays. Quantification was performed using calibration curves of peak areas versus alkaloid concentration for available authentic standards. Compounds not reliably detected in full scan mode were analyzed by multiple-reaction monitoring using the

1:1,000 diluted latex samples and parameters listed in Appendix 9-10. For compounds not

available as authentic standards, reported values represent relative abundance. Compounds were

identified or annotated based on retention time and CID spectrum compared with authentic

standards (Appendix 1; Appendix 5A/B) or reference data (Dolejš and Hanuš, 1967; Schmidt et

al., 2005; Schmidt et al., 2007; Wickens et al., 2006; Farrow et al., 2012; Desgagné-Penix et al.,

2012).

2.12.5 Chapter 5: (S) and (R)-Reticuline Percentage

VIGS samples analyzed by LC-MS in Chapter 5 were also analyzed by chiral HPLC-UV to

determine the percentage of (S)- and (R)-reticuline. Briefly, total (R,S)-reticuline from each

sample was isolated using a Silica Gel 60 F254 thin-layer chromatography plate (TLC, EMD

Chemicals, Gibbstown, New Jersey, USA; www.emdchemicals.com) and a solvent system of

acetone, toluene and ammonium ethanol (45:45:10). Reticuline spots were manually removed

from the TLC plate and extracted in 1mL of ethanol. Filtered samples (0.22µm PVDF filter)

were dried in a speed-vacuum concentrator and resuspended in 40µL ammonium bicarbonate

supplemented with 0.1% (v/v) diethylamine for chiral HPLC-UV analysis. Each sample (20µL) was injected onto a LUX cellulose-1 column, and (S)- and (R)-reticuline was separated using an

isocratic mix of 80% ammonium bicarbonate with 0.1% DEA (Solvent A) and 20% acetonitrile

(Solvent B). The elution of (S)- and (R)-reticuline was monitored at 284nm. The percent (S)

and (R)-reticuline was determined using the following calculations:

% (R)-reticuline = (R)-reticuline ACs ÷ (R)-reticuline ACs + (S)-reticuline ACs x 100

% (S)-reticuline = (S)-reticuline ACs ÷ (R)-reticuline ACs + (S)-reticuline ACs x 100

ACs=area counts

2.13 LC-MS Analysis of Papaverine and Pacodine

Papaverine and pacodine levels in different organs of the opium poppy chemotype

Veronica were determined by HPLC–MS/MS. Opium poppy tissues were flash frozen and

ground to a powder under liquid nitrogen using a TissueLyser II (Qiagen) at 30Hz for 1min with

a 30mL grinding jar and a 10mm stainless steel ball. Ground plant material was lyophilized and

extracted with 30mL acetonitrile per gram dry weight. Extracts were incubated for 16h at -20°C

and subsequently centrifuged at 4°C and 14000g for 10min to remove insoluble debris. The supernatant was transferred to a new tube and evaporated to dryness under vacuum. Samples were reconstituted in 10mM ammonium acetate (pH 5.5) containing 5% (v/v) acetonitrile, and

10μL were injected onto a Hypersil Gold C18 HPLC column (1.8μm, 2.1mm x 50mm; Thermo),

and compounds were separated using a gradient elution of 10mM ammonium acetate (pH 5.5)

containing 5% (v/v) acetonitrile (Solvent A) and acetonitrile (Solvent B). The flow rate was 500

μL.min-1 and the gradient was started at 0% solvent B, which was increased linearly to 7.5%

(v/v) by 1.5min and then immediately to 25% by 1.6min. Solvent B was increased linearly to

33% by 5min, 92% by 7min, and 98% by 7.6min. At 8.6min solvent B was reduced to initial

conditions by 8.7min for 4min of re-equilibration. Analytes were subjected to multiple reaction

monitoring using transitions established for papaverine (m/z 340.2 > 202.1 and 340.2 > 324.1)

and pacodine (m/z 326.1 > 188.1 and 326.2 > 310.1) and quantifications were performed using a

calibration curve of authentic papaverine.

2.14 Phylogenetic Analysis

Amino acid alignments were performed using ClustalW2 software (Chenna, 2003; Larkin

et al., 2007) and phylogenetic relationships were visualized using a bootstrapped phylogenetic

tree generated using the geneious 6 software package (Biomatters, Aukland, New Zealand) using

the Jukes-Cantor genetic distance model with neighbour-joining. The bootstrapped analysis was

performed with 1000 iterations to evaluate the statistical significance of phylogenetic tree nodes.

2.15 Genbank Accession Numbers

2.15.1 Accession Numbers of ODDs Sequences Used in this Thesis

AtACO1, Arabidopsis thaliana 1-aminocyclopropane-1-carboxylate oxidase 1

(NM_127517); AtF3H, Arabidopsis thaliana flavanone 3-hydroxylase (AY116957); AtFLS,

Arabidopsis thaliana flavonol synthase 5 (NM_001037054); AtGSLOH, Arabidopsis thaliana

putative ODD (AY114055); AtJRG21, Arabidopsis thaliana putative leucoanthocyanidin

dioxygenase (AJ298225); AtSRG1, Arabidopsis thaliana senescence-related gene 1 (X79052);

CjNCS1, Coptis japonica norcoclaurine synthase 1 (AB267398); CmGA20ox, Cucurbita

maxima gibberellin 20-oxidase (U61385); CmGA7ox, C. maxima gibberellin 7-oxidase

(U61386); CrD4H, Catharanthus roseus desacetoxyvindoline 4-hydroxylase (U71605);

DmH6H, Datura metel desacetoxyvindoline 4-hydroxylase (AF435417); HnH6H, Hyoscyamus

niger hyoscyamine 6β-hydroxylase (DQ812529); HvIDS2, Hordeum vulgare iron deficiency-

specific clone 2 (BLYIDS2NK); HvIDS3, H. vulgare iron deficiency-specific clone 3

(AB024058); MdACCO, Malus x domestica ACC oxidase (DQ137848); ObF7ODM1, Ocimum

basilicum flavone 7-O-demethylase (KM507365); Os_uncharacterized1, Oryza sativa putative

ODD (NM_001049521); Os_uncharacterized2, O. sativa putative ODD (CB663889); PcFS,

Petroselinum crispum flavone synthase (AY230247); PhF3H, Petunia hybrida flavanone 3 hydroxylase (X60512); PhFLS, Petunia hybrida flavonol synthase (Z22543); PsCODM, Papaver somniferum codeine O-demethylase (GQ500141); PsT6ODM, Papaver somniferum thebaine 6

O-demethylase (GQ500139); PsP7ODM, Papaver somniferum papaverine 7-O-demethylase

(KT159979); PsPODA, Papaver somniferum protopine O-dealkylase (GQ500140); PsDIOX4,

Papaver somniferum putative ODD (KC854329); PsDIOX5, Papaver somniferum putative ODD

(KC854330); PsDIOX6, Papaver somniferum putative ODD (KC854331); Pt_XP002300453,

Populus trichocarpa putative ODD (XM_002300417); Rc_EEF42734, Ricinus communis flavonol synthase/flavanone 3-hydroxylase (XM_002519715); SlACCO, Solanum lycopersicum ethylene-forming enzyme (NM_001246999); Ss_uncharacterized1, Citrus sinensis putative ODD

(CV886305); St_uncharacterized1, Solanum tuberosum putative ODD

(PGSC0003DMT400018663*); Tc_uncharacterized1, Theobroma cacao putative ODD

(Thecc1EG029575t1*); Vv_uncharacterized1, Vitis vinifera putative ODD (XM_002269051);

ZmBX6, Zea mays benzoxazin 6 (NM_001111630); ZmFLS/F3H, Z. mays flavonol

synthase/flavanone 3-hydroxylase (EU972786). The search tags for sequences marked with an

asterisk are available at http://phytozome.jgi.doe.gov/pz/portal.html.

2.15.2 Accession Numbers of AKR Sequences Used in this Thesis

Genbank accession numbers of sequences of AKRs used in this study are: PsCOR1.1–

PsCOR1.4; Papaver somniferum codeinone reductase 1.1–1.4 (AF108432, AF108433,

AF108434, AF108435); PsCOR2.1, Papaver somniferum codeinone reductase 2.1 (AF108438);

EcMECGOR, Erythroxylum coca methylecgonone reductase (E7C196); AtAKR, Arabidopsis

thaliana aldo-keto-reductase family 4 member C8 (NP_565871); MsCHR, Medicago sativa chalcone reductase (AAB41556).

2.15.3 Accession Numbers of CYP Sequences Used in this Thesis

PsCYP82Y1, Papaver somniferum N-methylcanadine-1-hydroxylase (AFB74617);

PsCYP82X1, Papaver somniferum 1,13-dihydroxy-N-methylcanadine-8-hydroxylase

(AFB74614); PsCYP82X2, Papaver somniferum 1-hydroxy-N-mnethylcanadine 13-hydroxylase

(AFB74616); PsCYP82N4, Papaver somniferum methyltetrahydroprotoberberine 14

monooxygenase (L7X3S1); PsCYP82N3, Papaver somniferum protopine 6-monoxygenase

(L7X0L7); EcCYPN2v2, Eschscholzia californica protopine 6-monoxygenase (F2Z9C1);

EcCYP82B1, Eschscholzia californica (S)-N-methylcoclaurine 3’-hydroxylase (AAC39454);

AtCYP82G1, Arabidopsis thaliana polypeptide 1 (NP_189154); AtCYP82C2, Arabidopsis thaliana polypeptide 2 (NP_194925); NtCYP82E4v1, Nicotiana tabacum cytochrome P450 monoxygenase (ABA07805); GhCYP82D1, Gossypium hirsutum cytochrome P450 monoxygenase (AII31758); ObCYP82D33, Ocimum basilicum cytochrome P450 monoxygenase

(AGF30364); MpCYP82D62, Mentha x piperita cytochrome P450 monoxygenase (AGF30366).

2.16 Gene Expression Analysis in Different Plant Organs

Organs of the opium poppy var. ‘Veronica’ were flash frozen in liquid N2 before excision.

Total RNA was extracted from each organ using the CTAB RNA extraction protocol (Meisel et al., 2005) and converted to cDNA using Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s protocol. Gene expression analysis was performed using qRT-

PCR on six biological replicates and three technical replicates using the primers listed in

Appendix 3B. Reference genes for qRT-PCR were elongation factor-1a (ELF-1a) and polyubiquitin 10 (UBQ10, Chapter 4), and actin, ELF-1a and UBQ10 (Chapter 5). Results for reference genes in chapters 4 and 5 yielded essentially identical results for all target genes.

Results of Chapter 4 are presented using ELF-1a and UBQ10 as the reference gene. Wherase results of chapter 5 are presented using ELF-1a as the reference gene. Efficiencies for all primer sets were approximately equal and always >90%. Relative transcript levels were determined using the 2-ΔΔCt method (Livak and Schmittgen, 2001) and statistical differences between sample groups were determined using a non-paired, two-way t-test.

Chapter Three: Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy

Summary

In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed

by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). This chapter further investigates the biochemical functions of

CODM and T6ODM as well as the functionally unassigned paralog DIOX2. These investigations revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and DIOX2, renamed protopine O-dealkylase

(PODA), showed novel and efficient O-dealkylation activities, including regio- and substrate- specific O-demethylation and O,O- demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is

catalyzed by CYPs in mammals. Preferred substrates for O,O-demethylenation by CODM and

PODA were protopine alkaloids that serve as intermediates in the biosynthesis of

benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to

suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological

role for these enzymes in the metabolism of protopine alkaloids, and revealed their indirect

involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.

3.1 Introduction

Benzylisoquinoline alkaloids (BIAs) are a large and structurally diverse group of nitrogenous specialized metabolites mainly produced in a restricted number of plants in the order

Ranunculales, including the family Papaveraceae. Opium poppy (Papaver somniferum) produces several BIAs many of which have important pharmacological properties, including the narcotic analgesics morphine and codeine, the cough suppressant and promising anticancer drug noscapine, the vasodilator and antispasmodic papaverine, and the antimicrobial agent sanguinarine (Hagel and Facchini, 2013). BIA biosynthesis begins with the conversion of two molecules of L-tyrosine to dopamine and 4-hydroxyphenylacetaldehyde, which undergo Pictet-

Spengler condensation to yield (S)-norcoclaurine (Samanani et al., 2004), the central intermediate to an estimated 2,500 alkaloids. Successive 6-O-methylation, N-methylation, 3’ hydroxylation, and 4’-O-methylation reactions result in the formation of the central branch-point intermediate (S)-reticuline. Intramolecular carbon-carbon and carbon-oxygen coupling of (S) reticuline and downstream pathway intermediates generate a plethora of structural backbones that are differentially modified through oxidation, reduction and the addition of various functional groups. In opium poppy, major BIA subgroups include 1-benzylisoquinoline (e.g. papaverine), morphinan (e.g. morphine), phthalideisoquinoline (e.g. noscapine), and benzo[c]phenanthridine (e.g. sanguinarine) alkaloids. A restricted number of enzyme families and superfamilies are known to be involved in BIA metabolism, including oxidoreductases such as CYPs and FAD-dependent oxidases (Hagel and Facchini, 2013). Recently, a non-biased comparative genomics approach revealed the involvement of an additional oxidoreductase family, namely 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODDs), in morphinan alkaloid metabolism (Hagel and Facchini, 2010a). Thebaine 6-O-demethylase (T6ODM) and codeine O demethylase (CODM) are ODD enzymes responsible for the penultimate step in codeine biosynthesis, and the final conversion of codeine to morphine, respectively. Prior to their discovery, the longstanding assumption was that morphinan alkaloid O-demethylation in opium

poppy was achieved by CYPs based on the participation of such enzymes in human morphine

metabolism (Zhu, 2008). T6ODM and CODM represent two of only a small number of plant enzymes capable of catalyzing O-demethylation. Moreover, the recruitment of these two highly similar enzymes represents an apparently isolated evolutionary event that conferred the unique ability to synthesize codeine and morphine to opium poppy.

The discovery of T6ODM and CODM provided a rational basis for hypothesizing the occurrence of additional O-demethylation events in BIA metabolism, and prompted a reevaluation of established biosynthetic pathways (Hagel and Facchini, 2010b). Such consideration revealed several alkaloid end products with functional group substitution patterns that suggested the O-demethylation of upstream pathway intermediates. For example, numerous protoberberine and aporphine alkaloids lack one, or both, methyl groups presumably derived from the 6-O and 4’-O-methyl moieties of the central intermediate (S)-reticuline. The existence of several alkaloids with such landmark substitution patterns suggests that O-demethylation could play a widespread role in BIA biosynthesis. The original isolation and characterization of

T6ODM and CODM targeted their roles in morphine biosynthesis. However, the possible relevance of these enzymes in other aspects of BIA metabolism has not yet been considered.

In my thesis, I extend the catalytic functions of T6ODM and CODM in opium poppy beyond the biosynthesis of morphine and assign an activity to the previously uncharacterized enzyme DIOX2, which was renamed protopine O-dealkylase (PODA). All three enzymes differentially accepted, in some cases with considerable catalytic efficiency, BIA substrates belonging to various structural subgroups. Certain protopine and protoberberine alkaloids were particularly effective substrates, in addition to the previously reported acceptance of morphinan alkaloids (Hagel and Facchini, 2010a). Interestingly, although several substrates undergo

regiospecific O-demethylation similar to the late steps in morphine biosynthesis, CODM and

PODA catalyze the O,O-demethylenation of methylenedioxy bridges on protopine alkaloids and,

in the case of PODA, on protoberberine alkaloids containing this functional group. The

metabolic roles of ODDs in the O-demethylation and O,O-demethylenation of protopine

compounds and downstream derivatives (Figure 3.1), such as sanguinarine and rhoeadine

alkaloids, was demonstrated by suppressing specific gene transcript levels using virus-induced

gene silencing (VIGS). The removal and perceived reformation of O-linked methyl groups and

methylenedioxy bridges appears to play a substantial role in the direction of flux though highly

branched BIA metabolism especially beyond the formation of the protoberberine backbone, from

which the protopine, benzo[c]phenanthridine, and rhoeadine subgroups are derived. Although

CYP-mediated O,O-demethylenation of amphetamine analogues (Fukuto et al., 1991; Meyer et

al., 2008; Meyer et al., 2009) and BIAs (Paul et al., 2004; Liu et al., 2009) has been reported in

mammalian systems, this is the first reported occurrence of O,O-demethylenation in plants and

the only one in which ODDs are involved. Plant ODDs perform a variety of reactions, including

hydroxylation of aliphatic or aromatic moieties, dehydration, O- and N-demethylation, γ-ring

formation, and oxidative removal of methylsulfinyl groups (Hagel and Facchini 2010b). My

thesis extends the function of ODDs to include O,O-demethylenation, and provides evidence for an expanded role of ODDs in BIA metabolism.

Figure 3.1. Biosynthesis of major protopine alkaloids and sanguinarine in opium poppy. Establishment of a methylene bridge in (S)-reticuline by the berberine bridge enzyme (BBE) yields the protoberberine alkaloid (S)-scoulerine, which can undergo O-methylation, incorporation of up to two methylenedioxy bridges, and N-methylation yielding quaternary alkaloids suitable for the formation of the protopine backbone via 14-hydroxylation. Subsequent 6-hydroxylation and tautomerization yields the benzo[c]phenanthridine alkaloid dihydrosanguinarine, which is further oxidized to sanguinarine. Abbreviations, CFS, cheilanthifoline synthase; SPS, stylopine synthase; TNMT, tetrahydroberberine N methyltransferase; MSH, N-methylstylopine 14-hydroxylase; PSH, protopine-6-hydroxylase; DBOX, dihydrobenzophenenthridine oxidase.

3.2 Results

3.2.1 Phylogenetic Analysis of Opium Poppy ODDs

Three novel and full-length cDNAs encoding previously uncharacterized ODDs (DIOX4,

DIOX5, and DIOX6) with substantial amino acid sequence identity to T6ODM, CODM, and

PODA were isolated from opium poppy. Alignment of opium poppy ODD amino acid sequences with a variety of characterized and uncharacterized plant ODDs showed several absolutely conserved residues, including those forming a canonical HXDX nH catalytic triad presumably required for coordinating Fe(II) and an RXS motif implicated in 2OG binding. A phylogenetic tree (Figure 3.2) derived from this alignment showed high bootstrap support for two ODD subclades, one containing T6ODM, PODA, CODM, and DIOX6 and another consisting of DIOX4 and DIOX5, distinct from other plant ODDs. Interestingly, the most similar plant proteins are all functionally uncharacterized.

Figure 3.2. Rooted neighbor-joining phylogenetic tree for opium poppy ODDs and others reported from plants. Bootstrap frequencies for each clade are percentages of 1000 iterations. Accession numbers are listed in Chapter 2. Abbreviations: AtF3H, Arabidopsis thaliana flavanone 3-hydroxylase; AtFLS, Arabidopsis thaliana flavonol synthase 5; AtGSOH, Arabidopsis thaliana putative ODD; AtJRG21, Arabidopsis thaliana putative leucoanthocyanidin dioxygenase; AtSRG1, Arabidopsis thaliana senescence-related gene 1; CjNCS1, Coptis japonica norcoclaurine synthase 1; CmG20O, Cucurbita maxima gibberellin 20 oxidase; CmG7O, Cucurbita maxima gibberellin 7-oxidase; CrD4H, Catharanthus roseus desacetoxyvindoline 4-hydroxylase; HnH6H, Hyoscyamus niger hyoscyamine 6 β-hydroxylase; MdACCO, Malus x domestica ACC oxidase; Os_uncharacterized1, Oryza sativa putative ODD; Os_uncharacterized2, Oryza sativa putative ODD; PcFS, Petroselinum crispum flavone synthase; PhF3H, Petunia x hybrida flavanone 3-β-hydroxylase; PhFLS, Petunia x hybrida flavonol synthase; PsCODM, Papaver somniferum codeine O-demethylase; PsT6ODM, Papaver somniferum thebaine 6-O-demethylase; PsPODA, Papaver somniferum protopine O,O demethylenase; PsDIOX4, Papaver somniferum putative ODD; PsDIOX5, Papaver somniferum putative ODD; PsDIOX6, Papaver somniferum putative ODD; Pt_XP002300453, Populus trichocarpa putative ODD; Rc_EEF42734, Ricinus communis flavonol synthase/flavanone 3 hydroxylase; SlACCO, Solanum lycopersicum ethylene-forming enzyme; Ss_uncharacterized1, Citrus sinensis putative ODD; St_uncharacterized1, Solanum tuberosum putative ODD; Tc_uncharacterized1, Theobroma cacao putative ODD; Vv_uncharacterized1, Vitis vinifera putative ODD; ZmBX6, Zea mays 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside dioxygenase; ZmFLS/F3H, Zea mays flavonol synthase/flavanone 3-hydroxylase.

3.2.2 Biochemical Characterization of Opium Poppy ODDs

Six soluble, His-tagged ODDs from opium poppy were produced in E. coli and purified using Co2+-affinity chromatography. All recombinant enzymes exhibited a molecular weight consistent with the theoretical masses of the predicted translation products. Each ODD was screened for enzymatic activity with one of 26 BIAs exhibiting a wide variety of structural backbones and functional group modifications. Full-scan LC-MS detected two different modifications – loss of either 12 or 14 amu – in the m/z of reaction products compared with the corresponding substrates incubated overnight with certain ODDs. No other reaction products were detected. DIOX4 and DIOX5 failed to convert any tested BIA to a detectable product, and only trace levels of products reduced by 14 amu were detected when DIOX6 was incubated overnight with (S)-reticuline or (S)-scoulerine. In contrast, T6ODM, CODM, and PODA converted a substantial number of tested BIAs to products reduced by either 12 or 14 amu

(Figure 3.3-3.4). Protopine and morphinan alkaloids showed the highest apparent turnover rates among potential substrates belonging to a variety of BIA structural categories. Since the efficient acceptance of protopine alkaloids was unexpected and the reaction products included compounds reduced by both 12 and 14 amu, the biochemistry of these conversions was investigated in more detail. Incubation of T6ODM, CODM, or PODA with allocryptopine resulted in products reduced by 14, 12, and 12 amu, respectively (Figure 3.3A). Incubation of

CODM or PODA with cryptopine yielded products reduced by 12 and 14 amu, respectively, whereas T6ODM catalyzed trace O-demethylation (Figure 3.3B). Finally, incubation of PODA with protopine reduced the m/z of the substrate by 12 amu and, again, T6ODM catalyzed no reaction (Figure 3.3C). CODM also reduced the m/z of protopine by 12 amu, but two distinct peaks at m/z 342 were detected (Figure 3.3C). Since no authentic standards were available, O demethylation and O,O-demethylenation reaction products were identified through interpretation 70

of CID spectra (Figure 3.5). O-Demethylation of allocryptopine by T6ODM was characterized

by a reaction product with a quasi-molecular ion ([M+H]+) reduced by 14 amu compared to the

substrate (Figure 3.5A-B), whereas O,O-demethylenation by PODA and CODM resulted in a

quasi-molecular ion reduced by 12 amu (Figure 3.5A-D). Comparison of CID spectra for

allocryptopine and these reaction products confirmed the reactions that occurred. The

allocryptopine standard (Figure 3.5A) and the product of assays containing allocryptopine and

T6ODM (Figure 3.5B) produce a typical even-numbered, a-type fragment ion at m/z 206 consisting of the isoquinoline moiety and oxygen at C-1 (Schmidt et al., 2007). The a-type ion can lose water to form the b-type ion at m/z 188. For products of assays containing allocryptopine and CODM or PODA (Figure 3.5C-D) the a-type ion lost 12 amu (m/z 194) resulting from loss of the alkyl on the methylenedioxy bridge of the substrate. This a-type ion can lose water to yield the b-type ion, which is also reduced by 12 amu (m/z 176) compared with the equivalent ions (Figure 3.5A-B). Parallel interpretations were applied to the reaction products in PODA or CODM assays containing cryptopine or protopine. CODM was the only enzyme capable of the O,O-demethylenation of either the A- or B-rings. Triple-quadrupole

MS/MS strongly suggested O-demethylation and O,O-demethylenation of allocrytopine and other alkaloids. However, since authentic standards were not available and compound quantities were insufficient to perform NMR, reaction product identification was supported by high- resolution MS and MS2 analysis using an LTQ-Orbitrap XL. Allocryptopine had a measured mass of 370.164461, corresponding to an elemental formula of C21 H24 NO5 (-0.782 ppm error).

The O-demethylation product of T6ODM and allocryptopine had a measured mass of 356.14926,

corresponding to an elemental formula of C20 H22 NO5 (0.030 ppm error). The O,O

demethylenation product of CODM or PODA and allocryptopine had a measured mass of

358.16486, corresponding to an elemental formula of C20 H24 NO5 (-0.11 ppm error). These

values support the initial identification of substrates and reaction products obtained by triple

quadrupole MS, but for further confirmation these compounds were subjected to MS2 exact mass

analysis. Allocryptopine yielded characteristic (Schmidt et al., 2007) isoquinoline product ions

at 206.08102 and 188.07040 m/z corresponding to the elemental formulae C11 H12 NO3 (-0.872 ppm error) and C11 H10 NO2 (-1.091 ppm error) consistent with the identification of the substrate.

The T6ODM reaction product of allocryptopine yielded the same characteristic isoquinoline fragments as the substrate 206.08102 and 188.07042 m/z, corresponding to elemental formulae

C11 H12 NO3 (-0.727 ppm error) and C11 H10 NO2 (-0.984 ppm error), which is consistent with a

B-ring O-demethylation of the substrate. The CODM reaction product of allocryptopine yielded

characteristic isoquinoline fragments at 194.08098 and 176.07042 m/z corresponding to

elemental formulae of C10 H12 NO3 (-0.978 ppm error) and C10 H10 NO2 (-1.051 ppm error),

which is consistent with an A-ring O,O-demethylenation of the substrate.

Figure 3.3. LC-MS extracted ion chromatograms of enzyme assays using CODM, T6ODM, or PODA with the protopine alkaloids (A) allocryptopine, (B) cryptopine, or (C) protopine as substrates showing either O-demethylation or O,O demethylenation. Loss of 14 atomic mass units indicates O-demethylation, whereas loss of 12 atomic mass units corresponds to O,O demethylenation. The figure indicates when no reaction was detected.

Figure 3.4. Relative activity, reaction catalyzed, and regiospecificity of T6ODM, CODM, and PODA with different benzylisoquinoline alkaloids as substrates. Values represent the mean percent maximum activity ± standard error of 4 independent replicates for each compound compared with the substrate showing the highest turnover, which was set to 100%. Trace activity indicates less than 1% turnover. For O-demethylcryptopine, R1=CH3 and R2=H, or R1=H and R2=CH3. Abbreviation: n.d., not detected.

Figure 3.5. Identification of ODD reaction products using allocryptopine as the substrate by collision-induced dissociation mass spectrometry. (A) Fragmentation of allocryptopine yielded a characteristic isoquinoline product ion at m/z 206 (a-type ion), and loss of water from m/z 206 yielded m/z 188 (b-type ion). (B) Fragmentation of the reaction product at m/z 356 from assays with T6ODM and allocryptopine yielded the same characteristic isoquinoline product ions indicating that O-demethylation occurred on the B-ring. Fragmentation of the reaction product at m/z 358 from assays with (C) PODA or (D) CODM and allocryptopine yielded the same characteristic isoquinoline product ions as the allocryptopine standard, but less 12 atomic mass units (m/z 194 and 176) indicating that O,O-demethylenation occurred on the A-ring.

3.2.3 Substrate range

The percent maximum conversion of T6ODM was highest with the morphinans oripavine

(100%) and thebaine (97%), but modest activity was also detected with the protoberberine (R,S)

canadine (32%). Conversion of the protopine allocryptopine (10%) and the 1-benzylisoquinoline

papaverine (2%) were relatively low. Trace O-demethylation activities below 1% were detected

with a small number of other 1-benzylisoquinoline, protoberberine, and protopine substrates

(Figure 3.4). CODM catalyzed the 3-O-demethylation of the morphinans codeine (90%) and

thebaine (81%), but showed the highest turnover rate with protopine (100%). Other protopines –

allocryptopine (50%), cryptopine (37%), and O-demethylcryptopine (41%) – were also accepted by CODM with modest efficiency for A- or B-ring O,O-demethylenation, depending on the position of the methylenedioxy bridge. The 1-benzylisoquinoline (S)-reticuline (16%), the aporphine isocorydine (6%), and the protoberberines (S)-scoulerine (70%) and (R,S) tetrahydropalmatine (17%) also exhibited low to relatively efficient conversion rates (Figure

3.4). Previously, no enzymatic function was detected for PODA (DIOX2) using a relatively limited range of substrates (Hagel and Facchini, 2010a). However, several BIA substrates were either O-demethylated or O,O-demethylenated by PODA (Figure 3.4). The most efficient O

demethylation reactions were observed with cryptopine (100%), (R,S)-tetrahydropalmatine

(14%) and (R,S)-tetrahydropapaverine (2%), but papaverine, thebaine, and oripavine were also

O-demethylated on the A-ring at trace levels. The most efficient substrate for O,O

demethylenation by PODA was protopine (79%) followed by allocryptopine (40%). Several

protoberberines with methylenedioxy bridges, including (R,S)-canadine (38%), berberine (19%),

and (R,S)-stylopine (16%) were also converted to corresponding hydroxyl-containing

derivatives. For all reactions involving protopine, protoberberine, and 1-benzylisoquinoline,

PODA displayed regiospecificity for the A ring, except for (R,S)-tetrahydropalmatine, which was

O-demthylated on the B ring. Several tested BIAs with O-linked methyl groups and/or methylenedioxy bridges were not accepted as substrates, including (+/-)-pavine the benzo[c]phenenthridines dihydrosanguinarine and sanguinarine, the phthalideisoquinolines narcotoline and noscapine, the aporphines (S)-isocorydine, (S)-boldine, (S)-corytuberine, (S) glaucine, and (S)-isothebaine, and the bisbenzylisoquinoline berbamine.

3.2.4 Reaction kinetics

CODM exhibited a Km value of 198 ± 47 µM with (S)-scoulerine as the substrate,

whereas PODA displayed a Km value of 27 ± 15 µM with cryptopine (Figure 3.6; Table 3.1).

Approximate Vmax values of 4.2 ± 0.7 pkat for PODA and 11.6 ± 1.4 pkat for CODM were

calculated using (S)-scoulerine and cryptopine, respectively, as substrates. The catalytic rate and

catalytic efficiency were 0.095 s-1 and 1291.4 s-1 M-1, respectively, for PODA and 0.034 s-1 and

480.1 s-1 M-1, respectively, for CODM. The insolubility in aqueous solution at saturating

concentrations (>500 µM) precluded a reliable Km determination for crytopine as a substrate of

CODM. Kinetic constants for other protopine alkaloids could not be measured owing to

insufficient availability of allocryptopine and the insolubility of protopine in aqueous solution at

saturating concentrations.

Figure 3.6. Steady-state enzyme kinetics of purified recombinant (A) PODA and (B) CODM using cryptopine and (S)-scoulerine, respectively, as substrates. Incubation time (45 min) and protein concentration (100 ng µl-1) were optimized prior to enzyme kinetic analyses. Values represent mean product formation (µM s-1) ± standard deviation of three independent measurements. Maximum velocity (Vmax ) and substrate affinity (Km), catalytic rate (Kcat ), and catalytic efficiency (Kcat /Km) were determined based on Michaelis‐Menten kinetics.

3.2.5 Reaction mechanics

The mechanisms of O-demethylation and O,O-demethylenation were investigated using a

fluorescence-based Nash assay for formaldehyde detection, and a coupled reaction with formate

dehydrogenase for formic acid detection. As expected, the production of formaldehyde as the

result of O-demethylation (Hagel and Facchini 2010a) showed a direct increase in response to

enzyme quantity in assays containing CODM and codeine (Figure 3.7A), or PODA and cryptopine (Figure 3.7B). In contrast, formaldehyde was not detected in assays containing

CODM and cryptopine (Figure 3.7A) highlighting a potentially important difference between the O-demethylation and O,O-demethylenation reaction mechanisms. The lack of formaldehyde

release in this case was comparable to incubating PODA with codeine, where no reaction

product was expected (Figure 3.7B). Limited substrate solubility and/or availability precluded

an examination of O,O-demethylenation catalyzed by PODA. Production of formic acid was not

detected in assays containing any substrate and enzyme combination.

Figure 3.7. Formaldehyde is a byproduct of the O-demethylation, but not the O,O demethylenation of benzylisoquinoline alkaloids. (A) Formaldehyde production increased in response to the amount of CODM used in assays containing codeine, which undergoes O demethylation, but formaldehyde was not detected in assays containing cryptopine, which undergoes O,O-demethylenation. (B) Formaldehyde production increased in response to the amount of PODA used in assays containing crytopine, which undergoes O-demethylation, but formaldehyde was not detected in assays containing codeine, which is not a substrate.

3.2.6 Virus-Induced Gene Silencing

Mining of an Illumina-based transcriptome library of opium poppy cultivar Bea’s Choice,

which was used for VIGS analysis, revealed expression of T6ODM and CODM at levels

comparable with other BIA biosynthetic genes. In contrast, transcripts encoding PODA were not

detected in the Bea’s Choice cultivar. Therefore, gene-silencing experiments were limited to

T6ODM and CODM using the previously described vectors pTRV2-T6ODM and pTRV2

CODM, respectively (Hagel and Facchini, 2010a). T6ODM and CODM transcript levels were

suppressed simultaneously using pTRV2-DIOX. Mature opium poppy plants infiltrated as

seedlings with mixed cultures of A. tumefaciens harbouring pTRV1 and pTRV2, the latter as an

empty vector or containing fragments of T6ODM or CODM, were screened for the occurrence of

viral coat protein transcripts by RT-PCR. For each utilized pTRV2 construct, six plants that

tested positive for viral coat protein transcripts (Figure 3.8A) were analyzed by qRT-PCR for

mean relative abundance of T6ODM and CODM transcripts compared with empty pTRV2 vector

as the control. As described previously (Hagel and Facchini, 2010a), plants exposed to A.

tumefaciens harbouring pTRV2-DIOX resulted in a significant suppression of both T6ODM and

CODM transcript levels (Figure 3.8B). In contrast, plants exposed to A. tumefaciens harbouring

pTRV2-T6ODM resulted in the significant suppression of only T6ODM transcript levels, whereas plants exposed to A. tumefaciens harbouring pTRV2-CODM resulted in the significantly reduced abundance of only CODM transcripts (Hagel and Facchini, 2010a). Plants with suppressed T6ODM and/or CODM transcript levels showed a significant alteration in the accumulation of several alkaloids. As reported previously (Hagel and Facchini, 2010a), reduced transcript levels of T6ODM alone, or T6ODM and CODM together, resulted in a substantial decrease in the accumulation of codeine and morphine, and an increase in the accumulation of thebaine (Figure 3.9A). In contrast, suppression of only CODM transcript levels resulted in a 83

significant increase in the accumulation of codeine at the expense of reduced levels of morphine

and oripavine. Unexpectedly, sanguinarine accumulation in the roots of plants with reduced

levels of T6ODM and/or CODM transcripts was also significantly lower (Figure 3.10A).

Although less pronounced, noscapine and papaverine levels were also reduced in plants with

reduced levels of T6ODM and/or CODM transcripts. In contrast, accumulation of the protopine

alkaloids cryptopine, protopine, and allocryptopine increased significantly and in several cases

substantially in response to the suppression of T6ODM and/or CODM transcript levels (Figure

3.10A). The less abundant protopine — O-demethylcryptopine — also increased in abundance

in CODM-silenced plants, but was unaffected in plants in which T6ODM transcript levels were

reduced. Similarly, accumulation of the protoberberines N-methystylopine and N methylcanadine was also elevated in response to the suppression of T6ODM and/or CODM transcript levels (Figure 3.10B). Interestingly, stylopine (Figure 3.9B) and canadine (Figure

3.10A) levels were unaffected or, in some cases, reduced in T6ODM- and/or CODM-silenced plants. Accumulation of other protoberberines showed variable effects in response to the suppression of T6ODM and/or CODM transcript levels. With a few exceptions, cheilanthifoline, tetrahydrocolumbamine, and tetrahydropalmatine levels were unaffected, whereas sinactine accumulation increased significantly in all cases (Figure 3.9B). Levels of the rhoeadine N methylporphyroxine (Figure 3.10B) also increased significantly in T6ODM- and/or CODM- silenced plants, whereas the related compounds papaverubine B and glaudine (Figure 3.9B) were more abundant only in some cases. Accumulation of the 1-benzylisoquinoline laudanosine also decreased in response to the suppression of T6ODM and/or CODM (Figure 3.9B).

Figure 3.8. Virus-induced gene silencing of T60DM, CODM, and PODA in opium poppy. (A) Detection of viral coat protein transcripts by RT-PCR in stem segments from within 10 mm of flower buds harvested from six independent plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 construct. (B) Relative CODM and T60DM transcript levels in stem segments from within 10 mm of flower buds harvested from six independent plants infiltrated with Agrobacterium tumefaciens harboring pTRV1 and the indicated pTRV2 construct. Values represent the mean± standard deviation. Statistically significance differences (P < 0.01) relative to values for the empty pTRV2 vector (*) were calculated using Student's t test.

Figure 3.9. Effect of virus-induced gene silencing on the accumulation of selected benzylisoquinoline alkaloids using pTRV2 constructs designed to suppress the transcript levels in opium poppy of all ODDs (pTRV2-DIOX), T6ODM (pTRV2-T6ODM), or CODM (pTRV2-CODM) compared with empty vector controls. Values represent the mean ± standard deviation of 6 independent replicates. (A) Morphinan alkaloids for which authentic standards are available allowing quantitative determination of alkaloid content. (B) Other compounds annotated using reference spectra for which only relative abundance could be determined. Statistical significance (*) at P < 0.01 was calculated using Student’s t test.

Figure 3.10. Effect of virus-induced gene silencing on the accumulation of selected benzylisoquinoline alkaloids using pTRV2 constructs designed to suppress the transcript levels in opium poppy of all ODDs (pTRV2-DIOX), T6ODM (pTRV2-T6ODM), or CODM (pTRV2-CODM) compared with empty vector controls. All alkaloids were isolated from latex except sanguinarine, which was extracted from roots. Values represent the mean ± standard deviation of 6 independent replicates. (A) Compounds for which authentic standards are available allowing quantitative determination of alkaloid content. (B) Compounds annotated using reference spectra for which only relative abundance could be determined. Statistically significance differences (P < 0.01) relative to values for the empty pTRV2 vector (*) were calculated using Student’s t test.

3.3 Discussion

The discovery of T6ODM and CODM as the first reported O-demethylases involved in

plant specialized metabolism provided rational support for the suggestion that O-demethylation is a widespread occurrence in BIA biosynthesis (Hagel and Facchini 2010a, Hagel and Facchini,

2010b). T6ODM was initially isolated using DNA microarrays and based on the differential analysis of transcripts found in high-morphine and morphine-free opium poppy cultivars.

DIOX2 (PODA) and CODM, which showed 85% and 72% amino acid sequence identity, respectively, with T6ODM were isolated through homology-based queries of opium poppy EST libraries. The targeted objective of previous work was to specifically identify the enzymes conferring the unique ability of opium poppy to produce codeine and morphine. The physiological roles of T6ODM and CODM as the enzymes catalyzing the antepenultimate and ultimate conversions in morphine biosynthesis were firmly established in opium poppy plants using VIGS (Hagel and Facchini 2010a). However, with such a focus on morphinan alkaloid metabolism only a limited number of potential alkaloid substrates were tested in vitro, and the analysis of alkaloid perturbations in T6ODM- and CODM-silenced plants was not comprehensive. Furthermore, roots were not analyzed in gene-silenced plants since they are not the major sites of morphinan alkaloid accumulation. However, opium poppy roots are a major site for the accumulation of the benzo[c]phenanthridine alkaloid sanguinarine. We previously noted that (S)-scoulerine is an effective substrate for O-demethylation by CODM, which was somewhat surprising owing to the structural dissimilarity of this protoberberine alkaloid compared with morphinan alkaloids. Moreover, a widespread role for O-demethylation was initially unexpected in the context of general BIA metabolism outside of morphine biosynthesis.

This result, combined with the occurrence of other BIA end products exhibiting substitution patterns indicative of O-demethylation events affecting upstream pathway intermediates, 91

suggested that ODDs were potentially multifunctional enzymes operating at numerous points

along the branched BIA biosynthetic network. My thesis pursued a more comprehensive

investigation of ODDs, which included an expanded and structurally diverse selection of

potential alkaloid substrates along with an enhanced detection system based on LC-MS for the

analysis of both enzyme assays and VIGS experiments. Such refined approaches not only

revealed that ODDs catalyze O-demethylation on a substantially broader array of substrates than

previously indicated (Hagel and Facchini, 2010a), but also facilitated the discovery of O,O

demethylenation as an entirely novel and unexpected activity in BIA metabolism.

The recent availability of deep transcriptome resources for opium poppy (Desgagné-Penix

et al., 2012) permitted the isolation of three new ODD homologues, DIOX4, DIOX5, and

DIOX6. Phylogenetic analyses revealed greater similarity between DIOX6, T6ODM, CODM,

and PODA compared with the more distant DIOX4 and DIOX5 (Figure 3.2). Catalytic functions for these new clones were not detected using a wide range of potential BIA substrates, although DIOX6 exhibited trace O-demethylase activity with (S)-reticuline and (S)-scoulerine.

Based on the apparent lack of activity on available BIAs, DIOX4, DIOX5, and DIOX6 were not subjected to VIGS analysis. Assays of recombinant T6ODM and CODM using morphinan alkaloid substrates were consistent with previous results (Hagel and Facchini 2010a), whereby strict regiospecificity was detected for either the 6-O-methyl (T6ODM) or 3-O-methyl (CODM) groups of thebaine, oripavine, and/or codeine (Figure 3.4). Although not reported previously

(Hagel and Facchini, 2010a), a trace level of 6-O-demethylase activity was detected for PODA with thebaine and oripavine. This discrepancy resulted from using a more sensitive and analytically accurate assay method compared to the earlier approach. Previously, routine assays

14 were carried out using an indirect method (Tiainen et al., 2005) whereby CO2 released via the

O-demethylation-coupled decarboxylation of [1-14C]-oxoglutarate was captured and measured by

scintillation counting. Spontaneous, uncoupled decomposition of [1-14C]-oxoglutarate in the

presence of recombinant enzyme preparations caused high background measurements, which

precluded the confident assignment of trace activities. In my thesis, the detection and

identification of reaction products by LC-MS, combined with the inclusion of a substantially

expanded array of BIA substrates, facilitated the discovery of several new catalytic functions

(Figure 3.4). The most striking result was the capacity of CODM and PODA to efficiently

catalyze the O,O-demethylenation of several compounds. In particular, protopine alkaloids, a

structural subclass of BIAs not previously considered, were among the best substrates together

with a variety of protoberberine alkaloids. PODA displayed a preference for O,O

demethylenation of the A-ring (isoquinoline moiety) of the protopine or protoberberine

backbone, whereas CODM targeted methylenedioxy bridges on either the A- or B-ring (benzyl

moiety) of only protopine alkaloids. Despite the extensive sequence similarity among opium

poppy ODDs only CODM and PODA were capable of catalyzing O,O-demethylenation on protopine and/or protoberberine substrates in vitro. Remarkably, T6ODM and PODA, which

display 85% amino acid sequence identity, catalyzed different reactions on the same substrate.

For example, (R,S)-canadine and allocryptopine were O-demethylated by T6ODM, but O,O demethylenated by PODA. The O,O-demethylenation activity of CODM was also restricted to protopine alkaloids, whereas PODA was also able to catalyze the O,O-demethylenation of

protoberberine substrates. BIAs containing methylenedioxy bridges, but belonging to the

benzo[c]penanthridine (e.g. sanguinarine) and the phthalideisoquinoline (e.g. noscapine)

structural subgroups were not accepted as substrates by any of the ODDs.

The high relative activity of CODM with respect to both the O,O-demethylenation of protopine and the O-demethylation of morphinan substrates (Figure 3.4) suggested an important role for both reaction types in opium poppy plants. Unfortunately, the relative insolubility of protopine and cryptopine and the limited availability of allocryptopine as the preferred protopine alkaloid substrates for O,O-demethylenation precluded the determination of kinetic constants for

CODM. However, the measured Km of CODM for the O-demethylation of (S)-scoulerine (198 ±

48 µM) (Table 3.1) was about 10-fold higher than the previously reported Km for codeine (21 ±

8 µM) as a substrate for O-demethylation (Hagel and Facchini 2010a) indicating that the enzyme

has a higher affinity for morphinan substrates. In contrast, PODA displayed a relatively high

affinity for cryptopine (27 ± 15 µM) as a substrate for O-demethylation, and kcat/Km comparisons

suggested that PODA is a more efficient enzyme than either CODM or T6ODM (Hagel and

Facchini 2010a).

Enzymatic O,O-demethylenation has not been reported frequently and no other plant

enzymes capable of catalyzing this reaction have been reported. CODM and PODA are the first

examples of ODDs in either plants or animals that catalyze O,O-demethylenation. In humans

various CYPs, including the general xenobiotic metabolizing enzyme CYP2D6, found in the

liver have been shown to O,O-demethylenate amphetamine analogues such as 3,4

methylenedioxy-methamphetamine (MDMA) commonly known as ‘ecstasy’ (Tucker et al.,

1994; Meyer et al., 2008; Meyer et al., 2009). Similarly, protopine and californine, a BIA

belonging to the pavine subgroup, are O,O-demethylenated by rat liver enzymes CYP2D1 and

CYP2C11 (Paul et al., 2004). O-Demethylation by ODDs, and probably by CYPs, proceeds via hydroxylation of the O-linked methyl group followed by the elimination of formaldehyde

(Purpero and Moran, 2007; Loenarz and Schofield, 2008). In agreement with known

mechanistic features of ODDs and previously acquired data (Hagel and Facchini 2010a), our

current Nash assay results indicated formaldehyde release associated with the O-demethylation of both codeine by CODM and cryptopine by PODA (Figure 3.7). Interestingly, the release of formaldehyde was not detected in association with the O,O-demethylenation of cryptopine by

CODM. Early investigations of the CYP-catalyzed O,O-demethylenation of MDMA suggested a mechanism involving formate ester hydrolysis, whereby the leaving group is formic acid (Fukuto et al., 1991; Lin et al., 1992). However, the release of formic acid was also not detected in association with the action of CODM on cryptopine indicating that the ODD-catalyzed O,O

demethylenation of BIAs proceeds via a distinct and as yet unknown mechanism.

The discovery of novel catalytic functions for recombinant T6ODM and CODM prompted

an expanded investigation of their physiological roles using VIGS, which has proven effective

for the characterization of numerous BIA biosynthetic genes in opium poppy (Hagel and

Facchini, 2010a; Desgagné-Penix and Facchini, 2012; Wijekoon and Facchini, 2012; Winzer et

al., 2012). PODA was not targeted for VIGS analysis owing to the lack of corresponding

transcripts in the Bea’s Choice cultivar. The PODA cDNA was isolated from a fungal elicitor-

treated opium poppy cell culture library (Zulak et al., 2007; Hagel and Facchini 2010a) and does

not display expression in the plant under normal physiological conditions. Expression of PODA

in response to fungal elicitor treatment suggests that the gene is inducible by certain

environmental factors. However, the lack of transcripts in the stem or root transcriptomes

indicates that PODA does not contribute to the basal BIA profile in opium poppy. The use of

LC-MS, including CID, as platform to analyze T6ODM- and/or CODM-silenced plants provided

an enhanced sensitivity and a robust metabolite identification capacity compared with previous

work, whereby only HPLC coupled with UV detection was used (Hagel and Facchini 2010a).

Broad-scope analysis of plants with reduced T6ODM and CODM transcript levels, compared

with controls, revealed remarkable, diverse, and unexpected effects on alkaloid phenotypes.

Suppression of T6ODM and/or CODM transcript levels affected morphinan alkaloid

accumulation as reported previously (Figure 3.9A) (Hagel and Facchini, 2010a). Briefly, suppression of T6ODM transcripts caused an increase in thebaine and oripavine levels and a corresponding decrease in codeine and morphine levels. In contrast, suppression of CODM caused an increase in codeine accumulation at the expense of morphine and oripavine. Extended analysis showed that similarly profound changes also occurred in protopine alkaloid levels, which significantly increased in response to the silencing of T6ODM, CODM, or both genes simultaneously (Figure 3.10A). The accumulation of protopine alkaloids in CODM-silenced plants was generally in agreement with the catalytic functions and substrate range of recombinant CODM, which showed high relative activity with protopine, allocryptopine, cryptopine and O-demethylcryptopine (Figure 3.4). However, the accumulation of these compounds in response to T6ODM-silencing was unexpected since recombinant T6ODM exhibited only trace to relatively minor activity with some protopine alkaloids tested as substrates. Off-target silencing of CODM in T6ODM-silenced plants was ruled out (Hagel and

Facchini, 2010a). However, it is possible that an additional, as yet uncharacterized ODD with

CODM activity was co-silenced. Alternatively, recombinant T6ODM might display different catalytic properties compared with the native plant enzyme. In this regard, 2D gel electrophoretic analysis of opium poppy latex suggests the occurrence of multiple charge isoforms of T6ODM and/or CODM (Decker et al., 2000), which might reflect the extensive post- translational modification of ODDs represented by only singular known transcripts.

Consequently, the catalytic functions of native ODDs might differ from the detected activities of

the corresponding recombinant enzymes (Liu and Dixon, 2001).

Interestingly, levels of papaverine and noscapine, which are regarded as metabolic end

products in opium poppy (Hagel and Facchini 2013) were reduced in silenced plants. It is

conceivable that the substantial increases in the accumulation of protopines and other alkaloids

reduced flux through other branch pathways requiring common upstream intermediates such as

(S)-reticuline. However, the reduction in sanguinarine levels was remarkable since

benzo[c]phenanthridine biosynthesis involves protopine as a key pathway intermediate (Figure

3.11). Unexpectedly, the elevated protopine level in silenced plants did not cause an increase in

sanguinarine accumulation, but instead was associated with a substantial decrease in

sanguinarine content in roots (Figure 3.10A). Possible explanations include the existence of

unknown regulatory mechanisms governing benzo[c]phenanthridine metabolism, such as transcriptional responses to elicitation by small molecules, or substrate inhibition of enzymes.

Coordinate transcriptional regulation of sanguinarine biosynthesis has been noted in opium poppy cell cultures (Zulak et al. 2007), affecting enzymes such as berberine bridge enzyme, tetrahydroprotoberberine N-methyltransferase (Liscombe and Facchini, 2007), and N methylstylopine hydroxylase (Beaudoin and Facchini, 2013) (Figure 3.1). The expression characteristics of the gene encoding protopine 6-hydroxylase (P6H) has not been characterized in opium poppy, and the biochemical aspects of protoberberine, protopine, and benzo[c]phenanthridine pathway regulation have not been investigated. However, substrate inhibition of P6H resulting from the elevated cellular pool of protopine or related alkaloids could potentially result in the decreased accumulation of sanguinarine. Substrate inhibition has been

reported for salutaridine reductase involved in morphine biosynthesis in opium poppy (Ziegler et

al., 2009) and is common in mammalian CYPs (Manoj et al., 2010; Tracy, 2006).

Silencing of T6DOM and/or CODM also resulted in elevated levels of N

methylporphyroxine (Figure 3.10B), whereas T6ODM-silenced plants accumulated more

glaudine compared with controls (Figure 3.9B). N-Methylporphyroxine and glaudine are

rhoeadine alkaloids, which are postulated to derive from protopine intermediates (Rönsch, 1977;

Montgomery et al., 1983; Tani and Tagahara, 1977). The elevated accumulation of rhoeadine

alkaloids could result from the increased availability of cryptopine (Figure 3.10A) and O

demethylcryptopine (Figure 3.10B) in silenced plants (Figure 3.11). The possible recycling of

protopine alkaloids must also be considered. For example, O-demethylation of allocryptopine by

T6ODM could potentially provide a mono-substituted methyl ether substrate for reformation of

the methylenedioxy bridge by relevant CYP enzymes (Ikezawa et al., 2007; Díaz Chávez et al.,

2011) (Figure 3.11). Similarly, the potential action of relevant O-methyltransferases (Morishige et al., 2002; Dang and Facchini, 2012) on of one of the two adjacent hydroxyl groups resulting from O,O-demethylenation would provide the required mono-substituted methyl ether moiety to reform the methylenedioxy bridge. Such recycling could regulate the cellular pool size of pathway intermediates, such as protopine, possibly involved in biochemical regulation via, for example, substrate inhibition.

O,O-Demethylenated benzo[c]phenanthridine, protoberberine, and protopine alkaloids have been detected as minor alkaloids in several members of the Papaveraceae including

Fummaria vaillantii (Ibragimova et al., 1974), Macleaya cordata (Lasskaya and Tolkachev,

1978), and Thalictrum javanicum (Bahadur and Shukla, 1983). Previously, the detection of O,O demethylenated BIAs was suggested as an artifact of extraction processes, although their natural

occurrence could not be discounted (Lasskaya and Tolkachev, 1978). Discovery of the O,O

demethylenation activity of PODA and CODM provides a biochemical basis for the natural

occurrence of such compounds in plants. However, O,O-demethylenated BIAs were not

detected in opium poppy plants subjected to VIGS suggesting their rapid recycling or

redistribution into other products, such as rhoeadine alkaloids.

In vitro profiling of recombinant enzyme activity supported by corresponding gene silencing in planta demonstrates that specific ODDs are extensively involved at multiple points in BIA biosynthesis. VIGS studies unequivocally demonstrated the widespread contribution of

T6ODM and CODM to BIA metabolism beyond the formation of morphine (Hagel and Facchini,

2010a), and suggested that the removal of O-linked methyl groups added early in the pathway plays a key role in the overall regulation of alkaloid biosynthesis. The discovery of new catalytic functions for ODDs, especially O,O-demethylenation, establishes a more complete appreciation for the complexities of BIA metabolism as a metabolic network with multiple interactive regulatory features, rather than the generally depicted linear routes (Hagel and Facchini, 2013).

Moreover, this data provokes important, interesting questions regarding the evolution of BIA metabolism. In particular, the apparently exclusive emergence of morphine biosynthesis in opium poppy (Hagel and Facchini 2010a) might have resulted from key mutations in ODDs originally participating in the formation of protopine, and indirectly, protoberberine,

benzo[c]phenanthridine, and rhoeadine alkaloids. Protopines and derivatives are taxonomically

more widely distributed than morphinan alkaloids (Kametani, 1968, 1974). In this context, the

substitution of only four amino acids in CODM resulted in the exclusive acceptance of codeine

for 3-O-demethylation, and the elimination of thebaine as a substrate (Runguphan et al., 2012).

As suggested (Hagel and Facchini, 2010b) previously, and despite the metabolic cost associated

with the extensive O-methylation of upstream BIA pathway intermediates, O-linked methyl groups and methylenedioxy bridges appear transient and replaceable in the context of the formation of end products, including morphine and, surprisingly, sanguinarine (Figure 3.11).

The catalytic functions of other opium poppy ODDs as well as homologues in plants related to opium poppy will provide more insight into the evolutionary origin and broad biochemical roles of ODDs in BIA biosynthesis.

100

Figure 3.11. Summary of enzymatic reactions catalyzed in vitro by T6ODM, CODM, and PODA using protopine alkaloid substrates and putative metabolic relationships with the benzo[c]phenanthridine sanguinarine and the rhoeadine alkaloids N-methylporphyroxine and glaudine in opium poppy. Compounds highlighted in yellow were detected and their levels were generally induced in plants subjected to virus-induced gene silencing resulting in the suppression of specific ODD transcripts. Levels of sanguinarine, highlighted in orange, were reduced in plants displaying lower ODD transcript abundance compared with controls. Enzymes in black were shown to catalyze the indicated conversion. Putative enzymatic reactions not tested empirically owing to a lack of substrate availability are shown in gray. Dashed arrows represent multiple, uncharacterized conversions. For O-demethylcryptopine, O-demethylallocryptopine, and O,O-demethylenated derivatives, R 1=CH3 and R2=H, or R1=H and R 2=CH3. Abbreviations: P6H, protopine 6-hydroxylase; DBOX, dihydrosanguinarine oxidase.

101

Table 3.1. Kinetic values for PODA and CODM with cryptopine and (S)-scoulerine, respectively

Km Vmax kcat kcat/Km Enzyme Substrate (µM) (pkat) (s-1) (s-1 M-1) PODA Cryptopine 27 ± 15 4.2 ± 0.7 0.034 1291.4 CODM (S)-Scoulerine 198 ± 48 11.6 ± 1.4 0.095 480.1

102

Chapter Four: Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe(II)-dependent dioxygenase from opium poppy

Disclaimer: This chapter presents the work that was done by SC Farrow and DC Burns. S.C.F. isolated and cloned P7ODM, performed all recombinant enzyme assays, mass spectrometric analyses, qRT-PCR experiments, and wrote the manuscript. D.C.B. conducted and interpreted the NMR analysis. The work is presented in full to facilitate understanding of the whole story.

Summary

Opium poppy (Papaver somniferum) produces several pharmacologically important BIAs including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic origin of pacodine and palaudine has not been established. As outlined in the previous chapter, three members of the ODD family in opium poppy display widespread O-dealkylation activity on several BIAs with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. This chapter expands on these discoveries and reports a novel ODD from opium poppy catalyzing the efficient substrate- and regio-specific 7

O-demethylation of papaverine yielding pacodine. The occurrence of papaverine 7-O demethylase (P7ODM) expands the enzymatic scope of the ODD family in opium poppy and suggests an unexpected biosynthetic route to pacodine.

4.1 Introduction

Opium poppy (Papaver somniferum) remains the sole commercial source for pharmaceutical opiates owing to a unique ability among plants to produce morphine and codeine

(Bernáth, 1998; Farrow et al., 2012). The plant also produces other BIAs with diverse pharmacological activities including the cough suppressant and promising anticancer agent noscapine, the antimicrobial sanguinarine, and the vasodilator and antispasmodic papaverine

(Hagel and Facchini, 2013).

103

Papaverine is tetra-O-methylated at the C6, C7, C3’ and C4’ positions (Figure 4.1). In

addition of O-linked methyl groups, the 1-benzylisoquinoline scaffold of papaverine contains a

3’-hydroxyl, and 1,2- and 3,4-desaturations yielding three fully conjugated rings compared with

(S)-norcoclaurine, the initial tri-cyclic intermediate from which all BIAs are derived. Whereas the biosynthetic pathways leading to morphine and noscapine have been largely elucidated

(Dang et al., 2014), the relatively less complex formation of papaverine has not been fully established (Winterstein and Trier, 1910; Brochmann-Hanssen et al., 1971; Brochmann-Hanssen et al., 1975; Han et al., 2010; Desgagné-Penix and Facchini, 2012; Hagel et al., 2012; Pathak et al., 2013). Two routes have been proposed; (i) the N-methyl pathway purports that papaverine is derived via the central branch-point intermediate (S)-reticuline, from which morphine and noscapine are also produced (Han et al., 2010), whereas (ii) the N-desmethyl pathway proposes that the upstream pathway intermediate (S)-coclaurine serves as the branch point to papaverine

(Brochmann-Hanssen et al., 1971; Brochmann-Hanssen et al., 1975; Desgagné-Penix and

Facchini, 2012; Pathak et al., 2013). Radioactive precursor labeling (Brochmann-Hanssen et al.,

1971; Brochmann-Hanssen et al., 1975), gene-suppression (Desgagné-Penix and Facchini, 2012) and comparative transcriptomics studies (Pathak et al., 2013) support the N-desmethyl pathway as the major route to papaverine.

Opium poppy also accumulates low levels of the papaverine analogues pacodine and palaudine, which lack O-linked methyl groups at C7 and C3’ respectively (Figure 4.1). The formation of pacodine and palaudine was logically proposed to proceed via the dehydrogenation of their corresponding tri-O-methylated precursors (S)-norcodamine and (S)-norlaudanine respectively (Brochmann-Hanssen et al., 1975). In support of this proposal, suppression of the gene encoding (S)-norreticuline 7-O-methyltransferase (N7OMT), which is responsible for the

104

substrate and regio-specific O-methylation of (S)-norreticuline yielding (S)-norcodamine, led to a

reduced accumulation of papaverine and pacodine in opium poppy (Desgagné-Penix and

Facchini, 2012). A flavoprotein-oxidase from opium poppy, named tetrahydropapaverine oxidase

(TPOX) in this context, was recently shown to dehydrogenate (S)-tetrahydropapaverine yielding

papaverine (Hagel et al., 2012). However, (S)-norlaudanine and (S)-norcodamine were not tested

as alternative substrates.

The characterization of ODDs catalyzing the widespread O-dealkylation of diverse BIAs in

opium poppy (Hagel and Facchini, 2010; Farrow and Facchini, 2013) prompted me to consider

the possibility that pacodine and palaudine were formed via the O-demethylation of papaverine.

My thesis reports the isolation and characterization of a novel ODD from opium poppy that

efficiently catalyzes the substrate- and regio-specific 7-O-demethylation of papaverine yielding pacodine.

105

Figure 4.1. Proposed biosynthesis of papaverine, pacodine and palaudine from (S) norcoclaurine in opium poppy showing conversions catalyzed by tetrahydropapaverine oxidase (TPOX) and papaverine 7-O-demethylase (P7ODM). Dashed arrows represent multiple enzymatic steps. The question mark indicates an unproven conversion.

106

4.2 Results

4.2.1 Isolation of DIOX7

The DIOX7 cDNA contained an open reading frame of 1110 nucleotides encoding a

polypeptide of 364 amino acids with a predicted molecular weight of 41.3 kDa (Figure 4.2).

DIOX7 contained the catalytic triad HX(D/E)XnH motif conserved among ODDs (Farrow and

Facchini, 2014) and the 2OG binding domain predicted from the crystal structure of Arabidopsis

thaliana leucoanthocyanidin dioxygenase (Wilmouth et al., 2002). DIOX7 shares considerable

sequence identity with previously characterized O-demethylases and O,O-demethylenases from

opium poppy: codeine O-demethylase (CODM; 75%), protopine O-dealkylase (PODA; 73%)

and thebaine 6-O-demethylase (T6ODM; 71%). The only other characterized ODD O demethylase is flavone 7-O-demethylase (F7ODM) from sweet basil (Ocimum basilicum) (Berim

et al., 2014), which shares 49% sequence identity with DIOX7.

Figure 4.2. SDS-PAGE and immunoblot analysis of purified recombinant P7ODM produced in Escherichia coli.

107

4.2.2 Expression and Characterization of DIOX7

Affinity purified, epitope-tagged DIOX7 was detected by SDS-PAGE and immunoblot analysis, and displayed an empirical molecular weight of approximately 41 kDa (Figure 4.2). In the presence of 2OG and other reaction components required by ODDs, DIOX7 converted papaverine (m/z 340) to a reaction product at m/z 326 (Figure 4.3A). The loss of 14 atomic mass units from papaverine (m/z 340) suggested that O-demethylation had occurred, which was supported by CID analysis of the reaction product (m/z 326) indicating the loss of an O-linked methyl group from the A-ring of papaverine (Figure 4.3B). Although the CID spectrum could not be used to determine whether O-demethylation occurred at C6 or C7, NMR analysis on the purified reaction product showed that DIOX7 was specific for the C7 O-linked methyl group, and the product was unequivocally identified as pacodine (Figure 4.3B; Appendix 6). Since no other alkaloids were accepted as substrates (Figure 4.4), DIOX7 was renamed papaverine 7-O demethylase (P7ODM).

108

Figure 4.3. O-demethylation of papaverine by P7ODM. (A) LC-MS-extracted ion chromatograms of enzyme assays using native or denatured P7ODM with papaverine (m/z 340) as the substrate showing O-demethylation and the formation of pacodine (m/z 326). (B) Identification of reaction product using papaverine as the substrate by collision-induced dissociation mass spectrometry showing the loss of a methyl group from the A-ring of papaverine.

109

Figure 4.4. Benzylisoquinoline alkaloids tested as potential substrates of P7ODM.

110

4.2.3 Biochemical Properties of P7ODM

P7ODM activity was stable across a broad temperature range from 0 to 25°C, but was

steadily inactivated along a linear temperature gradient from between approximately 30 and

55°C (Figure 4.5A). P7ODM also showed optimal activity between pH 8 and 9 in glycine buffer

(Figure 4.5B). Kinetic analysis of P7ODM was in agreement with the Michaelis-Menten model

-1 (Figure 4.5C) and exhibited a Km of 83 ± 13, a Vmax of 5.53 ± 0.38 picokatal, a Kcat of 0.09 s ,

-1 -1 and an enzymatic efficiency (Kcat/Km) of 1099 s M . Substrate or product inhibition was also

detected (Figure 4.5C). The kinetic values for P7ODM fall within the range of those calculated for T6ODM, CODM and PODA, and the presence of product or substrate inhibition appears to be a common feature among the characterized opium poppy O-demethylases (Hagel and

Facchini, 2010; Farrow and Facchini, 2013).

111

(μM)

Figure 4.5. In vitro characterization of affinity-purified P7ODM. (A) Temperature optimum. (B) pH optimum. (C) Steady-state enzyme kinetics. Values represent the mean ± SD of three replicates.

112

4.2.4 P7ODM Transcript Levels in Different Opium Poppy Organs

Relative P7ODM transcript levels were low in all organs, although stem showed lower abundance than roots and leaves (Figure 4.6A). Compared with CODM and T6ODM, transcript levels of P7ODM were substantially lower in all organs (Figure 4.6B).

4.2.5 Pacodine and papaverine levels in different opium poppy organs

Papaverine levels were more than three hundred-fold higher in all organs compared with pacodine (Figure 4.6C). Interestingly, both alkaloids showed a similar distribution profile with higher levels in flower buds, followed by stems, leaves and roots (Figure 4.6C). The accumulation of papaverine and pacodine were significantly (p<0.05) lower in root compared with aerial organs.

113

Figure 4.6. Relative abundance of P7ODM transcripts in opium poppy determined using qRT- PCR with elongation factor-1a and polyubiquitin 10 as the reference genes. Values represent the mean ± SD of six biological replicates. (A) Relative levels of P7ODM transcripts in different plant organs. Values with the same letter were not significantly different (p>0.05) as determined using a two-tailed, unpaired t-test. (B) Relative levels of P7ODM, T6ODM and CODM transcripts in different organs. (C) Papaverine and pacodine levels in different opium poppy organs. Values represent the mean ± SD of six biological replicates.

114

4.3 Discussion

The limited substrate acceptance of P7ODM with papaverine (Figure 4.3-4.4) is surprising considering the broad substrate range observed for T6ODM, CODM and PODA (Farrow and

Facchini, 2013), suggesting that P7ODM has more stringent requirements for substrate docking and coordination in the active site. As outlined in the previous chapter (Farrow and Facchini,

2013), T6ODM and PODA showed low and trace O-dealkylation activity with (R,S) tetrahydropapaverine and papaverine, respectively, indicating that minor amino acid substitutions between P7ODM and T6ODM/PODA facilitate the efficient O-dealkylation of papaverine by P7ODM. Insight into residues conferring stringency could be obtained through mutagenizing the regions of T6ODM and PODA thought to confer substrate specificity. A redesign of CODM has already been achieved by similar techniques (Runguphan et al., 2012), and when referencing the CODM sequence (Farrow and Facchini, 2014), substitutions to amino acids in non-conserved regions of P7ODM provide rational targets for understanding the O dealkylation of papaverine by P7ODM. The biosynthesis of pacodine and palaudine has been attributed to the aromatization of (S)-norcodamine and (S)-norlaudanine, respectively

(Brochmann-Hanssen et al., 1971; Brochmann-Hanssen et al., 1975; Desgagné-Penix and

Facchini, 2012). However, TPOX did not accept (S)-norreticuline (Hagel et al., 2012) suggesting that tetra-O-methylation of the papaveroline scaffold is required for dehydrogenation (Figure

4.1). The discovery of P7ODM is the first direct biochemical evidence for the formation of pacodine via the direct 7-O-demethylation of papaverine (Figure 4.1). Corroboration of the biochemical data (Figure 4.3; Figure 4.5) using virus-induced gene silencing (VIGS) was not possible owing to the low P7ODM transcript levels in opium poppy plants (Figure 4.6A-B).

However, the calculated Km of P7ODM is within the physiological concentration of papaverine

(Farrow and Facchini, 2013; Desgagné-Penix et al., 2012) and the calculated efficiency for the 115

reaction supports an activity for native P7ODM in papaverine O-demethylation. Furthermore, the

low concentration of pacodine (Figure 4.6C) is in agreement with the low P7ODM transcript abundance in all organs (Figure 4.6A) supporting a role for P7ODM in pacodine synthesis.

Interestingly, papaverine and pacodine accumulation show the same general profile in different organs (Figure 4.6C), which might correlate with the occurrence of P7ODM. It is not known whether TPOX (Figure 4.1) catalyzes the dehydrogenation of (S)-norcodamine to form pacodine; thus, dehydrogenation of (S)-norcodamine must also be considered as a possible route to pacodine.

The low P7ODM transcript levels in opium poppy plants (Figure 4.6A-B) suggest a potential requirement of conditional cues for gene expression. Biosynthetic genes involved in plant specialized metabolism are often inducible (van der Fits and Memelink, 2000; Cheong et al., 2002). Significantly, a massive induction in gene expression, including those encoding several BIA biosynthetic enzymes, is triggered by treatment of opium poppy cell cultures with a fungal elicitor (Zulak et al., 2007). Although cDNAs encoding P7ODM were not detected in the elicitor-induced opium poppy cell culture EST database (Zulak et al., 2007), alternative sources of induction might occur in the plant. The broad temperature optimum of P7ODM suggests that abiotic stressors could be required for P7ODM expression. In Arabidopsis thaliana, cold stress up-regulates several specialized metabolism genes including ODDs that operate in flavonoid and glucosinolate biosynthesis, and a ODD potentially involved in alkaloid biosynthesis (Hannah et al., 2005).

In general, the O-demethylation activity of P7ODM and related ODDs from opium poppy could be part of a transmethylation system involved in the maintenance of steady-state alkaloid levels (Poulton, 1981). As suggested for the biosynthesis of sanguinarine (Farrow and Facchini,

116

2013), O-dealkylation might also alleviate the accumulation of pathway intermediates that cause feedback regulation in BIA metabolism.

Papaverine has widespread uses in medicine including for the treatment of erectile

dysfunction (Bella and Brock, 2004), smooth muscle spasm (Liu and Couldwell, 2005), is a

cryo-preservative for blood vessels (Müller-Schweinitzer et al., 1993), is used in angioplasty and

coronary artery surgery (Takeuchi et al., 2004), and is a second line prophylaxis for migraine headaches (Koponen, 1978). Alternatively, the pharmacological properties of pacodine have not been reported. However, given the structural similarity to papaverine, pacodine or analogs thereof might exhibit similar or novel biological activities.

Previous phylogenetic analysis of ODDs from opium poppy and other species (Berim et

al., 2014) showed a close relationship between F7ODM from sweet basil and opium poppy

ODDs, which suggests a conservation of features required for O-dealkylation. With several

available ODDs to compare, elucidation of the structural basis for O-alkylation is possible. In a

broader context, the O-dealkylation of small molecules is considered rare in plant metabolism

(Berim et al., 2014), yet all plants contain several uncharacterized ODDs (Farrow and Facchini,

2014). A fundamental understanding of protein features conferring O-dealkylation reactions will

also allow prediction of O-dealkylation functions among uncharacterized ODDs including those

from other BIA producing plant species.

117

Chapter Five: Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy

Disclaimer: This chapter presents the work that was done by SC Farrow, JM Hagel, GAW Beaudoin and DC Burns. S.C.F. performed all recombinant enzyme assays, virus- induced gene silencing experiments and mass spectrometric analyses, and co-wrote the manuscript. J.M.H. constructed the yeast expression vectors, performed all qRT-PCR experiments, and co-wrote the manuscript. D.C.B. conducted and interpreted the NMR analysis. G.A.W.B. contributed to the initial gene isolations and VIGS vector construction. The work is presented in full to facilitate a more complete understanding of the story.

Summary The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. This chapter outlines the discovery of a fusion between a CYP and an AKR that catalyzes the S to R epimerization of reticuline via 1,2 dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and

Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent

CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2 dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinans.

An ancestral relationship is supported by a conservation of introns in gene fusions and independent orthologs. Suppression of REPI transcripts using VIGS in opium poppy reduced

(R)-reticuline and morphinan levels, and increased accumulation of (S)-reticuline and its O-

methylated derivatives. Discovery of REPI completes the isolation of genes responsible for

known steps of morphine biosynthesis.

5.1 Introduction

The pentacyclic morphinan alkaloids in opium poppy (Papaver somniferum) feature five

chiral carbons that limit the efficiency of chemical synthesis (Quasdorf and Overman, 2014). As

118

a result, agricultural production of opium poppy remains the sole commercial source for

pharmaceutical opiate production. Since the isolation of morphine in 1806, the intricate links

between opium poppy and the human condition have motivated extensive research on the

biosynthesis of morphinan alkaloids (Hagel and Facchini, 2013). Substantial progress toward

pathway elucidation was achieved during the 1960s, which supported a key hypothesis (Gulland

and Robinson, 1925) that morphine was derived via 1-benzylisoquinoline alkaloid

metabolism (Kirby, 1967). (S)-Reticuline emerged as the central 1-benzylisoquinoline intermediate, with its stereochemical inversion to (R)-reticuline purported as a pivotal gateway reaction since only the (R)-conformer could undergo subsequent phenol coupling to the morphinan scaffold (Battersby et al., 1965).

With the exception of the S to R epimerization of reticuline, all genes responsible for perceived conversions from dopamine and 4-hydroxyphenylacetaldehyde to morphine have been isolated (Figure. 5.1; Hagel and Facchini, 2013). The stereochemical inversion of (S)-reticuline

has been proposed to proceed via a 1,2-dehydroreticulinium cation intermediate, and two distinct

enzymes implicated in successive oxidation and reduction reactions have been at least partially

purified from opium poppy (Figure. 5.2). 1,2-Dehydroreticuline synthase (DRS) was partially

enriched and reported to convert (S)-reticuline to 1,2-dehydroreticuline in the absence of a

cofactor (Hirata et al., 2004), whereas 1,2-dehydroreticuline reductase (DRR) was purified to

apparent homogeneity and showed a strict requirement for NADPH (De-Eknamkul and Zenk,

1992). Although the genes encoding DRS and DRR have not been isolated, a potential clue to their identity was apparent from RNAi-mediated silencing of the codeinone reductase (COR) family in opium poppy. COR is an AKR operating several steps downstream of (S)-reticuline

(Figure. 5.1), yet silencing COR resulted in the accumulation of (S)-reticuline rather than

119

codeinone and morphinone (Allen et al., 2004). A possible explanation for this unexpected phenotype includes off-target gene silencing (Facchini and St-Pierre, 2005). My thesis reports the isolation and characterization of genes encoding reticuline epimerase (REPI), DRS and DRR from opium poppy and the common field poppy (Papaver rhoeas).

120

Figure 5.1. Complete morphine biosynthetic pathway in opium poppy. Abbreviations: TYDC, tyrosine/DOPA decarboxylase; TyrAT, tyrosine aminotransferase; NCS, norcoclaurine synthase; 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80B3, (S)-N-methylcoclaurine hydroxylase, 4’OMT2, 4’-O-methyltransferase 2; REPI, reticuline epimerase; CYP719B1, salutaridine synthase; SalR, salutaridine reductase; SalAT, salutaridinol acetyltransferase; T6ODM, thebaine 6-O-demethylase; COR, codeinone reductase; CODM, codeine O-demethylase.

121

Figure 5.2. Proposed two-step stereochemical inversion of (S)-reticuline to (R)-reticuline catalyzed by 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reducatse (DRR) in opium poppy. CYP719B1 converts (R)-reticuline to salutaridine, which undergoes a multistep transformation to morphine.

122

5.2 Results

5.2.1 COR Paralog Search Identifies CYP82Y2 in Opium Poppy

The possible off-target co-suppression of DRR transcripts in COR-silenced opium poppy plants prompted a search for COR paralogs (Allen et al., 2004; Facchini and St-Pierre, 2005).

Two COR paralogs were isolated, one of which showed an in-frame extension of the coding region upstream of the predicted AKR (PsDRR1) domain (Figure. 5.3A). The upstream

(PsDRS) domain annotated as a member of the CYP82 family and was designated CYP82Y2.

The entire coding region was amplified from opium poppy by RT-PCR ruling out an assembly artifact as the source of the fusion. An independent AKR (PsAKR2) displaying 84% identity to the PsDRR1 region of CYP82Y2 showed no evidence of fusion (Figure. 5.3A). Full-length orthologs encoding fusion proteins sharing 100 and 96% amino acid identity with opium poppy

CYP82Y2 were also identified in Papaver setigerum and P. bracteatum, respectively. In contrast, the most similar orthologs in Papaver rhoeas encoded independent CYP82 (PrDRS) and AKR (PrDRR) proteins sharing 76 and 86% amino acid identity with the respective domains of the opium poppy fusion (Figure. 5.3A). Phylogenetic analysis indicated a close relationship between DRS and other CYP82 family members involved in BIA biosynthesis (Figure. 5.4A), whereas DRR is related to AKR enzymes involved in cocaine biosynthesis (Jirschitzka et al.,

2012) and chalcone metabolism (Ballance and Dixon, 1995), in addition to several COR variants

(Figure. 5.4B). Isolation of the corresponding genes from opium poppy and P. rhoeas showed conservation in the approximate location of introns (Figure. 5.3B).

123

Figure 5.3. Maps of cDNAs and genes encoding reticuline epimerase (REPI), 1,2 dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reducatse (DRR). Abbreviations: Ps, Papaver somniferum; Pr, P. rhoeas. (A) Full-length cDNAs showing the location of DRS and DRR domains in the REPI fusion and the alignment of independent DRS and DRR orthologs. Black and white boxes represent open reading frames and linker sequences between DRS and DRR domains, respectively. (B) Structure of genes encoding PsREPI, PrDRS, PrDRR and PsAKR2. Boxes and solid lines represent exons and introns, respectively. bp, base pairs.

124

125

Figure 5.4. Unrooted phylogenetic trees for selected (A) cytochrome P450 monoxygenases in the CYP82 family and (B) NADPH-dependent aldo-keto reductases. Maximum- likelihood phylogenetic analyses were performed using the phyml program. Gene sequences were obtained from NCBI based on BLAST results for 1,2-dehydroreticuline synthase (CYP82Y2) and 1,2-dehydroreticuline reductase (DRR). Accession numbers are listed in Chapter 2. Abbreviations: PsCYP82Y1, Papaver somniferum N-methylcanadine-1-hydroxylase; PsCYP82X1, P. somniferum 1,13-dihydroxy-N-methylcanadine-8-hydroxylase; PsCYP82X2, P. somniferum 1-hydroxy-N-mnethylcanadine 13-hydroxylase; PsCYP82N4, P. somniferum methyltetrahydroprotoberberine 14-monooxygenase; PsCYP82N3, P. somniferum protopine 6 monoxygenase; EcCYPN2v2, Eschscholzia californica protopine 6-monoxygenase; EcCYP82B1, E. californica (S)-N-methylcoclaurine 3’-hydroxylase; AtCYP82G1, Arabidopsis thaliana polypeptide 1; AtCYP82C2, A. thaliana polypeptide 2; NtCYP82E4v1, Nicotiana tabacum cytochrome P450 monoxygenase; GhCYP82D1, Gossypium hirsutum cytochrome P450 monoxygenase; ObCYP82D33, Ocimum basilicum cytochrome P450 monoxygenase; MpCYP82D62, Mentha x piperita cytochrome P450 monoxygenase. PsCOR1.1–PsCOR1.4; Papaver somniferum codeinone reductase 1.1–1.4; PsCOR2.1, P. somniferum codeinone reductase 2.1; EcMECGOR, Erythroxylum coca methylecgonone reductase; AtAKR, Arabidopsis thaliana aldo-keto-reductase family 4 member C8; MsCHR, Medicago sativa chalcone reductase. Scale bar represents amino acid substitutions per site.

126

5.2.2 CYP82Y2 is Reticuline Epimerase

As defined below, the opium poppy CYP82Y2 fusion was named reticuline epimerase

(PsREPI), and corresponding CYP polypeptides from opium poppy and P. rhoeas (Figure. 5.3) were designated 1,2-dehydroreticuline synthase (PsDRS and PrDRS, respectively). Similarly, active AKR polypeptides from opium poppy and P. rhoeas (Figure. 5.3) were named 1,2 dehydroreticuline reductase (PsDRR1 and PrDRR, respectively). Constructs containing a CYP were co-expressed in Saccharomyces cerevisiae with a plant CPR. The following complete or partial polypeptides were tested for catalytic activity: (i) the CYP82Y2 fusion (pCPR/PsREPI),

(ii) the isolated CYP domain (pCPR/PsDRS), (iii) the isolated AKR domain (pPsDRR1), and (iv) the independent AKR (pPsAKR2) from opium poppy, and (v) the non-fused CYP82Y2

(pCPR/PrDRS) and (vi) AKR (pPrDRR) from P. rhoeas. The recombinant CYP and CPR proteins in S. cerevisiae microsomes were validated by immunoblot analysis (Figure. 5.5A).

His-tagged AKR domains (pPsDRR1, pPsAKR2 and pPrDRR) were expressed in Escherichia coli and purified by affinity chromatography (Figure. 5.5B; Figure 5.5C). Microsomal preparations or purified proteins were assayed using (S)-reticuline, 1,2-dehydroreticuline, or (R) reticuline as substrates. Under standard reaction conditions, PsREPI converted (S)-reticuline to

~5% 1,2-dehydroreticuline and ~10% (R)-reticuline (Figure. 5.6). When supplied with 1,2 dehydroreticuline PsREPI produced ~80% (R)-reticuline, but (R)-reticuline was not accepted as a substrate. PsDRS and PrDRS each converted (S)-reticuline to 1,2-dehydroreticuline, although the opium poppy CYP domain showed a higher turnover (>50%) and neither enzyme yielded (R) reticuline (Figure. 5.6). Using assay conditions favouring alkaloid substrate reduction, PsDRR1 and PrDRR both converted 1,2-dehydroreticuline to (R)-reticuline, although the opium poppy

AKR domain was substantially more efficient (Figure. 5.7A; Figure. 5.8; Table 5.1). Under

127

conditions favouring oxidation, PsDRR1 and PrDRR converted (R)-reticuline, but not (S)

reticuline, to 1,2-dehydroreticuline (Figure. 5.7B). PsAKR2 (Figure. 5.3) showed no activity with any tested substrate (Figure. 5.9).

128

Figure 5.5. Heterologous production and/or purification of recombinant enzymes. (A) Immunoblot analysis showing α-FLAG-tagged PsREPI, PsDRS and PrDRS, and α-c-Myc-tagged cytochrome P450 reductase (CPR) in microsomal fractions of Saccharomyces cerevisiae. Recombinant PsREPI, PsDRS and PrDRS were detected using α-FLAG antibodies, whereas recombinant CPR was detected using α-c-Myc antibodies. Each lane contained 2 μg of total microsomal protein. (B) Coomassie Blue-stained, denaturing polyacrylamide gel showing His- tagged PsDRR1, PrDRR and PsAKR2 (arrowhead) produced in Escherichia coli and purified using cobalt-affinity chromatography. (C) Immunoblot analysis showing recombinant, purified PsDRR1, PrDRR and PsAKR2 detected using α-His antibodies.

129

Figure 5.6. Catalytic functions of PsREPI, PsDRS and PrDRS using (S)-reticuline, 1,2-dehydroreticuline, and (R)-reticuline as substrates. (A) Extracted ion chromatograms generated by LC-MS showing the conversion of (S)-reticuline (m/z 330) to 1,2 dehydroreticuline (m/z 328) by PsREPI, PsDRS and PrDRS, and the formation of reticuline from 1,2-dehydroreticuline by PsREPI. (B) Chiral separation showed the formation of (R)-reticuline by PsREPI from either (S)-reticuline or 1,2-dehydroreticuline. All LC MS product peaks displayed identical quasi-molecular ions and collision-induced dissociation spectra compared with authentic standards (Appendix 1). No conversions were detected in negative control assays (pCPR). Data are representative of at least four independent replicates. For clarity, reaction substrates and products are highlighted in blue and orange, respectively.

130

\ Figure 5.7. Catalytic functions of PsDRR1 and PrDRR using (S)-reticuline, 1,2 dehydroreticuline, and (R)-reticuline as potential substrates. (A) Extracted ion chromatograms showing the conversion of 1,2-dehydroreticuline (m/z 328) to reticuline (m/z 330) by PsDRR1 and PrDRR in the presence of NADPH, as measured by LC-MS. (B) Extracted ion chromatograms showing the conversion of (R)-reticuline to 1,2-dehydroreticuline by PsDRR1 and PrDRR in the presence of NADP+, as measured by LC-MS. All product peaks displayed identical quasi-molecular ions and collision-induced dissociation spectra compared with authentic standards (Appendix 1). No conversions were detected in negative control assays using denatured enzymes. Data are representative of at least four independent replicates. For clarity, reaction substrates and products are highlighted in red and green, respectively.

131

Figure 5.8. Chiral separation and detection by HPLC-UV showed the formation of (R) reticuline by PsDRR1 and PrDRR from 1,2-dehydroreticuline. All product peaks displayed identical quasi-molecular ions and collision induced dissociation (CID) spectra compared with authentic standards (Appendix 1). No conversions were detected in negative control assays using denatured enzymes. Data are representative of at least four independent replicates.

132

Table 5.1. Kinetic data for PsREPI, PsDRS, PrDRS, PsDRR1 and PrDRR.

Km Vmax kcat kcat /Km Enzyme Substrate Product -1 -1 -1 (μM) (pkat) s (M s )

1,2 a PsDRS (S)-Reticuline 3.4 ± 0.5 0.157 ± 0.006 - - Dehydroreticuline

1,2 a PrDRS (S)-Reticuline 2.6 ± 0.9 0.031 ± 0.003 - - Dehydroreticuline

PsREPI (S)-Reticuline (R)-Reticuline 4.3 ± 0.6 0.09 ± 0.01a - -

PsDRR1 1,2-Dehydroreticuline (R)-Reticuline 13.2 ± 6.7 43.9 ± 5.5 39.16 2,977,757

PrDRR 1,2-Dehydroreticuline (R)-Reticuline 13.5 ± 2.2 1.08 ± 0.04 0.043 3,157

1,2 PsDRR1 (R)-Reticuline 34.9 ± 2.6 4.8 ± 0.1 4.26 122,121 Dehydroreticuline

1,2 PrDRR (R)-Reticuline 25.2 ± 3.4 0.49 ± 0.02 0.019 762 Dehydroreticuline a Microsomal preparations were used for CYP enzyme assays, precluding enzyme quantification and calculation of kcat and kcat/Km values.

Figure 5.9. PsAKR2 does not show the same function as PsDRR1. Extracted ion chromatograms showing that PsAKR2 failed to convert (S)-reticuline or (R)-reticuline to 1,2 dehydroreticuline in the presence of NADP+, and did not yield reticuline from 1,2 dehydroreticuline in the presence of NADPH. Data are representative of at least four independent replicates.

133

5.2.3 Requirement for N-methylated Substrates

PsDRS and PrDRS were assayed with various 1-benzylisoquinoline alkaloids as potential

substrates (Figure. 5.10) and only three were efficiently accepted: (S)-reticuline, (S)-3’-hydroxy

N-methylcoclaurine, and (S)-N-methylcoclaurine. PsDRS showed the highest turnover with (S) reticuline, whereas PrDRS was most active with (S)-N-methylcoclaurine and exhibited lower activity toward (S)-reticuline. Corresponding alkaloids lacking an N-methyl group were not accepted as substrates, and 1-benzylisoquinolines with C7 or C3’ O-linked methyl groups were not converted perhaps due to increased steric hindrance.

PsREPI also catalyzed the stereochemical inversion of (S)-N-methylcoclaurine (Figure

5.11). PsDRR1 and PrDRR reduced 1,2-dehydro-N-methylcoclaurine to N-methylcoclaurine

although the low product yield precluded determination of the enantiomer (Figure. 5.11).

PsREPI, PsDRS and PrDRS all showed a pH optimum of ~7.8 using (S)-reticuline as the

substrate. In contrast, reduction of 1,2-dehydroreticuline by PsDRR1 and PrDRR exhibited a

broad pH optimum between 6 and 9 (Figure. 5.12), and oxidation of (R)-reticuline by PsDRR1

and PrDRR was optimal at pH ~8.8. Using (S)-reticuline as the substrate, PsREPI, PsDRS and

PrDRS showed a relatively low Km of 4 µM compared with PsDRR1 and PrDRR, which showed

Km values of 13 µM for the reduction of 1,2-dehydroreticuline (Table 5.1; Figure. 5.13).

PsDRR1 and PrDRR showed higher catalytic efficiency for the reduction of 1,2

dehydroreticuline compared with the oxidation of (R)-reticuline, with PsDRR1 displaying

considerably more efficiency than PrDRR in the conversion of 1,2-dehydroreticuline to (R)

reticuline.

Two contaminants with m/z 344 in the 1,2-dehydroreticuline standard were also

transformed to compounds with m/z 346 in PsDRR1 and PrDRR assays (Figure. 5.14A; Figure

134

5.14B). The m/z 346 reaction products were subjected to structural elucidation by NMR

(Appendix 7) and identified as diastereomers of α-hydroxyreticuline, indicating that the two contaminants were enantiomers of α-hydroxy-1,2-dehydroreticuline (Figure. 5.14C; Figure

5.14D). Unfortunately, assignment of the enantiomers could not be confirmed.

Figure 5.10. Benzylisoquinoline alkaloids tested as potential substrates of 1,2 dehydroreticuline synthase. Values (PsDRS / PrDRS) represent percentage conversion relative to the substrate showing the highest turnover, which was set at 100%. Compounds named in blue are intermediates in morphine biosynthesis. nd, not detected.

135

Figure 5.11. Catalytic functions of PsREPI, PsDRS and PrDRS on (S)-N-methylcoclaurine as a substrate. (A) Extracted ion chromatograms showing the conversion of (S)-N methylcoclaurine (m/z 300) to N-methyl-1,2-dehydrococlaurine (m/z 298) by PsREPI, PsDRS and PrDRS. (B) Chiral separation and detection by HPLC-UV showed the formation of (R)-N methylcoclaurine by PsREPI from (S)-N-methylcoclaurine. No conversions were detected in negative control assays (pCPR). The (R)-N-methylcoclaurine product peak displayed identical quasi-molecular ions and collision-induced dissociation (CID) spectra compared with authentic (S)-N-methylcoclaurine (Appendix 1). The identity of N-methyl-1,2-dehydrococlaurine was inferred from the m/z 298 reaction product CID spectrum (Appendix 1). Furthermore, (C) the reaction product generated by the incubation of PsDRS and PrDRS with (S)-N-metylcoclaurine was consumed by PsDRR1 and PrDRR supporting the identification of m/z 298 as N-methyl-1,2 dehydrococlaurine. Data are representative of at least four independent replicates. For clarity, reaction substrates and products are highlighted in blue and orange, respectively.

136

Figure 5.12. pH optima of PsREPI, PsDRS, PsDRR1, PrDRS and PrDRR. PsREPI activity was determined for the conversion of (S)-reticuline to (R)-reticuline. PsDRS and PrDRS activity is based on the conversion of (S)-reticuline to 1,2-dehydroreticuline. PsDRR1 and PrDRR forward reactions were performed using 1,2-dehydroreticuline and NADPH as substrates, yielding (R)-reticuline. PsDRR1 and PrDRR reverse reactions were performed using (R)-reticuline and NADP+ as substrates, yielding 1,2-dehydroreticuline. The overall pH optimum of oxidation and reduction reactions involved in the conversion of (S)-reticuline to (R)-reticuline via 1,2-dehydroreticuline is indicated by the yellow highlight. The pH optimum of the oxidation converting (R)-reticuline to 1,2-dehydroreticuline is indicated by the orange highlight. Values are the mean ± standard deviation of three replicates.

137

Figure 5.13. Steady-state enzyme kinetics for PsREPI, PsDRS, PrDRS, PsDRR1 and PrDRR. (A, B, C) Recombinant enzymes in total microsomal protein extracts from Saccharomyces cerevisiae were assayed using different concentrations of (S)-reticuline (up to 64 µM) at a fixed concentration of NADPH (250 µM). (D, F) Recombinant, purified PsDRR1 and PrDRR from Escherichia coli were assayed using different concentrations of 1,2 dehydroreticuline (up to 300 µM) at a fixed concentration of NADPH (500 µM), or (E,G) using different concentrations of (R)-reticuline (up to 300 µM) at a fixed concentration of NADP+ (500 µM). All enzymes followed Michealis-Menten reaction kinetics with associated constants shown in Table 5.1. Assays were performed under linear product-formation conditions. For PsDRR1 and PrDRR, forward reactions were performed at pH 7, whereas reverse reactions were performed at pH 8.8. Kinetics for PsDRS, PrDRS and PrDRS were determined at pH 7.5. Values are the mean ± standard deviation of three replicates.

138

Figure 5.14. Function of PsDRR1 and PrDRR on contaminants in the authentic 1,2 dehydroreticuline standard. (A) Extracted ion chromatograms showing the conversion of two contaminants with m/z 344 to products with m/z 346 by native PsDRR1 and PrDRR in the presence of NADPH. NMR analysis showed that both m/z 346 products were stereoisomers of α hydroxyreticuline (Appendix 7). (B) Extracted ion chromatograms showing that the purified α hydroxyreticuline (m/z 346) stereoisomers were not converted back to α-hydroxy-1,2 dehydroreticuline (m/z 344) in the presence of NADP+. (C) Collision-induced dissociation spectra of α-hydroxy-1,2-dehydroreticuline and α-hydroxyreticuline. (D) Structures of α hydroxy-1,2-dehydroreticuline and α-hydroxyreticuline. The C1 chirality shown for α hydroxyreticuline was assumed based on the formation of (R)-reticuline by PsDRR. Data are representative of at least four independent replicates. For clarity, reaction substrates and products are highlighted in blue and orange, respectively.

139

5.2.4 Physiological Characterization of Reticuline Epimerase

VIGS was used to determine the physiological role of PsREPI in opium poppy (Figure.

5.15; Appendix 11-13). The pTRV2-REPI-5’ construct targeted the 5’-untranslated region and a short portion of the PsREPI coding region (Appendix 11). The pTRV2-COR1.1 contained

(Appendix 12) the fragment used previously for the RNAi-mediated silencing of the COR gene family in opium poppy (Allen et al., 2004), and the pTRV2-REPI-a construct targeted a corresponding region in the AKR domain of PsREPI (Appendix 13). The pTRV2-REPI-5’ construct was designed to test the physiological role of REPI, whereas the pTRV2-REPI-a and pTRV2-COR1.1 constructs were included to consider the possibility that the previously reported pathway block at (S)-reticuline (Allen et al., 2004) resulted from off-target suppression of REPI transcripts levels.

Relative REPI transcript abundance was significantly reduced in plants subjected to VIGS using all three constructs (Figure. 5.15A), and the resulting alkaloid profiles were remarkably similar in both the latex and roots (Figure. 5.15B–D; Appendix 11-13). A substantial increase in reticuline levels was accompanied by accumulation of the related O-methylated alkaloids codamine, laudanine and laudanosine (Figure. 5.15B). Notably, the ratio of (S)- to (R)-reticuline increased from approximately 3:1 in controls to almost 50:1 in REPI and COR co-silenced plants

(Figure. 5.15C; Appendix 11-13). Levels of morphine, codeine, and thebaine were substantially reduced, and consistently associated with a significant increase in the accumulation of noscapine, which is derived from (S)-reticuline. To ensure observed phenotypes did not result from off- target silencing, qRT-PCR was performed on all known morphine biosynthetic genes (Figure.

5.16). No off-target silencing effects were detected in plants subjected to VIGS using the pTRV2-REPI-5’ construct. In contrast, reciprocal suppression of REPI and COR transcripts was

140

observed using the pTRV2-REPI-a, and pTRV2-COR1.1 constructs owing to substantial (77%)

nucleotide sequence identity between PsDRR1 and COR1.1 (Appendix 14). Off-target silencing of SalR was observed using pTRV2-REPI-a and pTRV2-COR1.1 despite low nucleotide sequence identity with either REPI or COR1.1. Consistent with all known alkaloid biosynthetic

genes, REPI transcripts were most abundant in stems (Figure. 5.17).

141

Figure 5.15. Virus-induced gene silencing in opium poppy supports the role of PsREPI in morphinan alkaloid biosynthesis. (A) Relative REPI and COR transcript abundance in REPI-silenced (REPI-5’ and REPI-a) and COR-silenced (COR1.1) plants compared with controls (pTRV2). (B) Accumulation of reticuline and O-methylated derivatives in the latex of REPI- and COR-silenced plants compared with controls. (C) Relative abundance of S and R enantiomers of reticuline in REPI- and COR-silenced plants compared with controls. (D) Relative abundance of major alkaloids in the latex of REPI- and COR-silenced plants compared with controls. Values represent the mean ± standard deviation of 16 biological replicates. Asterisks represent significant differences determined using an unpaired, two-tailed Student t test (p <0.05).

142

Figure 5.16. Relative transcript abundance of benzylisoquinoline alkaloid biosynthetic genes in opium poppy in plants subjected to virus-induced gene silencing targeting PsREPI and PsCOR1.1. qRT-PCR was performed using primers listed in Appendix 4. The reference gene for results shown was Elf-1a. Values represent the mean ± standard deviation of six biological replicates. An unpaired, two-tailed Student t test (p <0.05) was used to define significant differences (asterisks) in transcript abundance between control (pTRV2) and PsREPI or PsCOR1.1-silenced plants.

143

144

Figure 5.17. Relative transcript abundance of benzylisoquinoline alkaloid biosynthetic genes in different opium poppy organs. qRT-PCR was performed using primers listed in Appendix 4. Relative transcript abundance is presented (A) for each biosynthetic gene in four different organs or (B) for each organ with respect to all biosynthetic genes. Values represent the mean ± standard deviation of six biological replicates.

145

5.3 Discussion

A fusion protein consisting of CYP and AKR domains catalyzes the S to R stereochemical

inversion of reticuline in opium poppy. The independent DRR purified from opium poppy

seedlings (De-Eknamkul and Zenk, 1992) could represent a member of the DRR family absent in

our stem transcriptome (Desgagné-Penix et al., 2012). Similarly, the previously detected oxidase

might also occur only in seedlings (Hirata et al., 2004). In addition to the lack of a cofactor

requirement, further evidence that the previously isolated oxidase was not a CYP82Y2 is

apparent in the reported substrate norreticuline, which is not accepted by PsDRS or PrDRS

(Figure 5.10). Of potential relevance, (S)-tetrahydroprotoberberine oxidase has been reported to yield dehydrobenzoisoquinolines from corresponding substrates including norreticuline (Amann and Zenk, 1987; Amann et al., 1988).

Dehydrogenation of (S)-reticuline by DRS represents a new reaction type in the CYP82 family. Other CYP82 members catalyze ring hydroxylations in benzylisoquinoline alkaloid biosynthesis (Takemura et al., 2013; Beaudoin and Facchini, 2013; Dang and Facchini, 2014;

Dang et al., 2014). Desaturation is not a common CYP function in plants, although sterol desaturases (Morikawa et al., 2006; Field and Osbourn, 2008) and desaturating flavone synthases

(Martens and Mithöfer, 2005) have been reported. CYP-mediated desaturation of sterols is not

thought to involve a hydroxylated intermediate (Mizutani and Sato, 2011), although

hydroxylation and subsequent dehydration are relevant in flavone formation (Akashi et al.,

1999). Similarly, DRS could catalyze the stereospecific hydroxylation of (S)-reticuline at C1,

followed by loss of water from the hemiaminal yielding the 1,2-dehydroreticulinium cation

intermediate with no apparent conversion to Z- and/or E-enamines. Equilibrium with enamine

isomers has been reported for 1,2-dehydroreticuline in aqueous solution greater than pH 8 (He

146

and Brossi, 1993). Although the acidic conditions used for LC-MS analyses would mask

enamines formed during REPI and DRS assays (Figure. 5.6), labeling studies have ruled out

enamine formation during the S to R transition of reticuline (Battersby et al., 1968) implying

tight coordination or shielding of the intermediate. The stereoselective hydrogenation of a

carbon-nitrogen double bond by DRR also represents a novel AKR reaction type. Most AKRs

catalyze the stereoselective reduction of aldehyde or carbonyl functional groups to primary or

secondary alcohols, respectively, although members of the AKR1D family (i.e. 5β-reductases)

catalyze irreversible steroid double-bond reductions (Penning, 2014).

Selective pressure on binding affinity was arguably greater for the CYP domain of PsREPI,

which is reflected in the lower Km of PsDRS compared with PsDRR1 (Table 5.1). However, the potential impact of a gene fusion on biosynthetic performance is not apparent solely from kinetic data. Enzyme fusions can contribute to increased product yield without substantial alterations in

Km or catalytic efficiency owing to the extreme proximity of active sites (Wang et al., 2011;

Brodelius et al., 2002). Efficient substrate channeling and coordination of the reaction intermediate through electrostatic interactions would further reduce selective pressure on the binding affinity of PsDRR1. Substrate channeling and domain-domain interactions have been reported for enzyme fusions involved in microbial folate metabolism (Sharma et al., 2013). In plants, domain-domain interactions are well-established features of bifunctional diterpene synthases, such as abietadiene synthase, which catalyzes two mechanistically distinct sequential reactions in separate active sites (Peters et al., 2003). Substrate channeling in PsREPI, combined with possible membrane-anchored proximity to salutaridine synthase (CYP719B1), could confer functional superiority over independent DRS and DRR enzymes. Moreover, coordination of the

1,2-dehydroreticuline cation intermediate would prevent tautomerization to an enamine (He and

147

Brossi, 1993). Metabolic engineering strategies using artificial protein fusions further underscore the value of integrating separate pathway enzymes into a single catalytic unit (Elleuche, 2014;

Marienhagen and Bott, 2013).

Performing VIGS using highly conserved regions of COR1.1 and REPI caused a reciprocal reduction in transcript abundance and precisely mimicked the alkaloid phenotype (Figure. 5.15;

Appendix 12-13) resulting from RNAi-based silencing of COR (Allen et al., 2004). In contrast,

VIGS performed using a REPI-specific region produced the same alkaloid phenotype without co suppression of COR transcript levels (Figure. 5.15; Appendix 11) confirming the physiological role of REPI. The increased accumulation of O-methylated 1-benzylisoquinolines was consistent with metabolic redirection of (S)-reticuline away from morphine biosynthesis. Reduced flux to morphine was also reflected in the elevated accumulation of noscapine, which is derived from

(S)-reticuline. Co-silencing of SalR (Figure. 5.16) was unexpected based on low nucleotide sequence identity with REPI and COR (Appendix 14), although off-target transcript suppression is sometimes associated with post-transcriptional gene silencing (Dang et al., 2014).

The stereochemical inversion of small molecules has been reported in amino acid metabolism, sugar degradation, cofactor repair, and natural product biosynthesis in plants and microbes, and is generally catalyzed by single enzymes (Pillai et al., 2006; Morrison et al., 2005;

Marbaix et al., 2011; Stapon et al., 2003). The evolution of a gene fusion combining CYP and

AKR catalytic functions has established a single-enzyme solution to a potential metabolic bottleneck in morphine biosynthesis. The existence of REPI also supports the suggestion that gene fusions provide evidence of functional interaction within metabolic pathways (Suhre,

2007).

148

Gene clusters are regarded as mechanisms of functional association within metabolic

pathways (Nützmann and Osbourn, 2014). Recently, a ten-gene cluster consisting of most noscapine biosynthetic genes was identified in opium poppy (Winzer et al., 2012), but the role of genetic linkage in the morphine branch pathway is not yet known. The REPI fusion might have arisen from a deletion between two tightly linked genes, combining the CYP and AKR domains into a single translation product. The exclusive detection of REPI in morphinan alkaloid- producing Papaver species suggests a relatively recent linkage event. Despite the isolation of independent orthologs encoding DRS and DRR in P. rhoeas, reports are rare on the accumulation of 1,2-dehydroreticuline or (R)-reticuline in plants. However, the relatively common occurrence of dimeric (R,S)-, (S,R)-, and (R,R)-bisbenzylisoquinolines (Schiff, 1991) suggests a potential physiological role for independent DRS and DRR enzymes. The correlation between morphinan alkaloid biosynthesis and the REPI fusion suggests that an efficient mechanism for (R)-reticuline production was required to sustain metabolic flux into the branch pathway. Interestingly, a search of the 1000 Plants database (Matasci et al., 2014) showed that P. rhoeas contains orthologs encoding proteins with 75–80% amino acid similarity to salutaridine synthase, salutaridine reductase and salutaridinol 7-O-acetyltransferase, which convert (R) reticuline to thebaine in opium poppy. Functional characterization of these homologs could reveal additional bottlenecks in morphinan alkaloid metabolism beyond potential improvements afforded by the REPI fusion.

149

Chapter Six: Overview, Future Perspectives and Conclusions

6.1 Overview of Thesis Research

6.1.1 2-oxoglutarate/Fe(II)-dependent Dioxygenases in BIA Metabolism

The late O-demethylation steps in morphine biosynthesis in opium poppy are catalyzed by the landmark ODDs CODM and T6ODM. The initial discovery of T6ODM and CODM focused on their role in morphine biosynthesis, wheras my thesis focussed on discovering additional roles for T6ODM and CODM in BIA metabolism. In addition, my thesis also investigated roles for the functionally unassigned ODD paralog DIOX2 as well as novel ODDs from opium poppy in

BIA metabolism. This was pursued in light of the observations that CODM efficiently catalyzed the O-demethylation of the structurally related BIA (S)-scoulerine (Hagel and Facchini, 2010a), and the occurrence of other BIAs in opium poppy exhibiting substitution patterns indicative of

O-demethylation activity (Hagel and Facchini, 2010b). Part of this research included an expanded and structurally diverse selection of potential BIA substrates along with an enhanced detection system based on LC-MS for the analysis of both enzyme assays and VIGS experiments. These state of the art approaches not only revealed that T6ODM and CODM catalyze O-dealkylation on a substantially broader array of substrates than previously indicated but also facilitated the discovery of new O-dealkylation activities for DIOX2 (PODA) and

DIOX7 (P7ODM). Furthermore, my comprehensive investigation of ODDs from opium poppy led to the discovery of O,O-demethylenation by CODM and PODA on certain BIAs containing methylenedioxy bridges. This is significant because O,O-demethylenation is an entirely novel and unexpected activity in BIA metabolism and plants. These discoveries will undoubtedly lead to a better understanding of BIA metabolism, and could help us understand the metabolism of other plant specialized metabolites.

150

6.1.2 Stereochemical Inversion of Reticuline by a P450 Oxidoreductase Fusion Protein in Opium Poppy

The RNAi-mediated silencing of the COR gene family in opium poppy led to the striking and unexpected accumulation of the upstream central pathway intermediate (S)-reticuline as opposed to COR empirical substrates (Allen et al., 2004). This phenotype was initially attributed to a negative feedback response initiated by silencing COR that affected relevant pathway genes/transport steps or the abolishment of a larger interdependent enzyme complex through the silencing of COR. Alternatively, my thesis considered that the accumulation of reticuline in opium poppy was the result of off-target gene silencing of a COR paralogue partaking in the stereochemical inversion of reticuline. By taking advantage of large transcriptome assemblies and using a combination of homology-based gene discovery, reverse genetics and state of the art analytical techniques, my thesis outlines the identification and characterization of an AKR/CYP fusion protein that is responsible for the two-step stereochemical inversion of reticuline in opium poppy. This discovery completes the discovery of all perceived steps leading to morphine in opium poppy and at the same time rationalizes the phenotype observed in the RNAi mediated silencing of COR in opium poppy (Allen et al., 2004). Before this discovery, natural fusion proteins were not known in plants, however, my thesis presents the first report of a plant fusion protein that appears to alleviate a potential pathway bottlenecks en route to morphine in opium poppy. This discovery will undoubtedly alter the current gene discovery approach, and could lead to the discovery of additional fusion proteins that are involved in the metabolism of other specialized metabolites. Furthermore, the discovery of the fusion protein has enabled the assembly of the entire morphine pathway in a microorganism (Galanie et al., 2015). This is significant because it could lead to an alternative opiate production platform that could help alleviate the global pain epidemic.

151

6.2 Future Perspectives

6.2.1 ODD O-dealkylases from Other Plant Species

A remaining question upon completion of my thesis is whether or not ODDs from other

BIA producing plant species are involved in BIA metabolism. A thorough search of BIAs

occurring in other plant species reveals those that are lacking O-methyl or methylenedioxy groups that were present on established or putative pre-cursors (Figure 6.1) providing some support for the occurrence of O-dealkylases in these pathways. Similarly, in chapter 3, a potential regulatory role was proposed for opium poppy ODDs in sanguinarine metabolism through the O,O-demethylenation of protopine alkaloids and the subsequent recycling or redistribution of these metabolites to other BIA pathways in an attempt to regulate flux.

Considering that protopines and sanguinarine are widely distributed among members of the

Papaveraceae, a hypothesis going forward is that orthologs of opium poppy ODDs have a conserved role in sanguinarine and protopine metabolism.

While writing my thesis, an ODD (F7ODM) was discovered from sweet basil that catalyzes the 7-O-demethylation of methoxylated flavones (Berim et al., 2014). O-dealkylation is considered rare in plant metabolism; however, this discovery, coupled with those described in my thesis, suggest that O-dealkylation reactions by ODDs may be more prevalent than originally thought. In this context, a phylogenetic tree presented in Berim et al., (2014) containing both uncharacterized and characterized ODDs illustrates the clustering of opium poppy, sweet basil and uncharacterized ODDs from functionally characterized ODDs, suggesting that sequences within this region have the requisite features for O-dealkylation reactions. With the availability of deep transcriptome libraries for numerous plant species (Xiao et al., 2013; Matasci et al.,

2014; Facchini et al., 2012), as well as state of the art molecular and analytical techniques, these questions can be addressed. Our recent transcriptome analysis of 20 BIA producing plant

152

species provides an excellent resource to pursue these ideas, as potential ODD candidates have

already been identified (Hagel et al., 2015).

Figure 6.1. Examples of BIAs with altered O-methylation patterns (right) relative to the established or putative precursors (left). Green highlighting indicates the position(s) of potential O-demethylation or O,O-demethylenation.

6.2.2 Potential Significance of Protein Fusions

The discovery of the CYP/AKR protein fusion catalyzing the stereochemical inversion of reticuline in opium poppy completes all perceived steps in morphine biosynthesis. Interestingly,

153

only morphinan containing species (i.e. Papaver bracteatum) were found to contain the fusion protein, while the taxonomically related Papaver rhoeas, which does not accumulate morphinan alkaloids, contains functionally equivalent but independent orthologs. As such, an objective going forward is to better understand the significance of the protein fusion for the synthesis of morphinan alkaloids. As suggested, the fusion protein could facilitate a more catalytically efficient inversion of (S)-reticuline to (R)-reticuline by substrate channeling and shielding of the dehydroreticuline intermediate from other potential enzymatic conversions. In this context, transformation of the morphinan-free P. rhoeas with PsREPI could help decipher the significance of the fusion protein for the production of morphinan alkaloids. Alternatively, comparing the localization of PsREPI in P.somniferum to PrDRS and PrDRR in P.rhoeas could reveal spatial and architectural features that help explain the significance of the protein fusion for the production of morphinan alkaloids in opium poppy. Finally, a thorough study on additional

DRR and DRS homologues from other BIA producing plant species could reveal unique features of the opium poppy fusion protein that enables the efficient epimerization of reticuline.

6.2.3 Characterization of Novel BIA Biosynthetic Genes

Although the enzymatic steps of some medicinally important BIA pathways have been elucidated in model plants like opium poppy, there are numerous BIA biosynthetic genes awaiting discovery. For example, the enzymes leading to rhoeadine alkaloids could involve a

CYP, carboxyl esterase, and O-methyltransferase whereas the spiro-isoquinoline, pavine and cularine alkaloids can be rationalized through C-C or C-O phenol coupling reactions that are likely catalyzed by CYPs. With the availability of several transcriptome libraries, functional genomics tools like VIGS and state of the art analytical equipment such as mass spectrometry and ultra-performance liquid chromatography, the elucidation of these pathways could be realized relatively quickly. 154

6.2.4 Regulation of BIA biosynthesis

There is a wealth of biochemical information regarding the enzymatic steps involved in

specialized metabolic pathways; however, a growing curiosity in the field is how these pathways

interact with those of primary metabolism. In Catharanthus roseus, terpenoid indole alkaloids

are regulated by the signalling hormone methyl jasmonate through the action of the ORCA3

transcription factor (van der Fits and Memelink, 2000). Whether similar mechanisms occur in

BIA producing plant species remains unknown. It has been demonstrated that topical plant

hormone applications affect the concentration of BIAs in opium poppy; however, the underlying

mechanisms causing these fluctuations are not known. Considering the lack of knowledge

linking primary and specialized metabolism for plants in general, elucidation of how primary

metabolism regulates BIA metabolism should be investigated. This could involve classical

hormone treatments in conjunction with metabolite analysis and RNA-seq or investigation of the

promoter regions of BIA biosynthetic genes for prediction of transcription factors.

6.2.5 Reconstitution of BIA Biosynthetic Pathways in Microorganisms.

The medicinal utility of BIAs have made them intriguing targets for microbial BIA-

pathway reconstitution with the gold standard being the reconstitution of the morphinan

biosynthetic pathway (Rathbone and Bruce, 2002). Incremental advancements from a small

number of molecular biochemistry laboratories (Facchini and De Luca, 1994; Pauli and Kutchan,

1998; Unterlinner et al., 1999; Morishige et al., 2000; Grothe et al., 2001; Choi et al., 2002;

Samanani et al., 2004; Ziegler et al., 2006; Gesell et al., 2009; Hagel and Facchini, 2010; Lee and Facchini, 2011; Farrow et al., 2015; Winzer et al., 2015) have made the reconstitution of the morphine biosynthetic pathway in Saccharomyces cerevisiae possible (Galanie et al., 2015).

The discovery of PsREPI described in my thesis and reported at the same time by a separate group (Winzer et al., 2015) filled the perceived void in morphinan biosynthesis and has

155

facilitated the complete synthesis of thebaine from tyrosine in yeast with a scaffold in place for de novo morphine synthesis (Thodey et al., 2014; DeLoache et al., 2015). While this assembly is a significant milestone in the metabolic engineering of opiates in microorganisms, the yields reported in this study are in the low microgram range, while a commercially competitive opiate yield would require at least a 200,000-fold improvement (Galanie et al., 2015).

Low yields highlight a significant hurdle to clear before microorganisms are a viable alternative to the current poppy-based opiate industry. In this context, morphinan alkaloids are derived from reductive pathways that are heavily dependent on NADPH, and as such, the system-wide redox balance could require optimization to fulfill the aim of large-scale opiate production in a microorganism (Kavšček et al., 2015). Similarly, if yeast is the desired chassis for BIA production, than overcoming aerobic ethanol fermentation is of critical importance for achieving commercially competitive yields of metabolites other than ethanol (Kavšček et al.,

2015). For the most part, this has been addressed by reducing glycolytic flux, and in one promising example replacing hexose transporters with a chimeric transporter significantly decreased ethanol production while at the same time increasing heterologous protein expression

(Ferndahl et al., 2010; Henricsson et al., 2005). While additional options such as codon optimization, protein engineering, promoter strategies and enzyme variants, etc. are available for optimizing product output in a microorganism (Trenchard and Smolke, 2015; Kavšček et al.,

2015), one cannot overlook having extensive knowledge of the native plant metabolic pathways for clues that could improve engineering efforts. In this respect, future bioengineering efforts should look to understand the important regulatory features that help streamline the production and accumulation of opiates in the plant. These may include transporters and additional enzymes that maintain a balance between BIA synthesis and catabolism that ensures the optimal

156

concentration of intermediates for the maintenance of pathway flux. As such, enzymology,

biochemistry and gene discovery will continue to be irreplaceable tools for a successful

microorganism-based opiate production platform.

The world health organization estimates that 83% of the world lives in countries with

limited or non-existent access to pain relief, and providing access to analgesia is among one of

its top initiatives (http://apps.who.int/medicinedocs/en/d/Js20982en/). In this regard, opiates are

considered among the best medicines for the treatment of severe pain, in pain management, and

end-of-life care. With the reconstitution of the morphine biosynthetic pathway in a microorganism recently disclosed, there is now a potential to provide restricted countries with opiate access. Although there are considerable social and political implications surrounding this technology, to put this technology into a positive light, a successful opiate production platform in microorganisms could go a long way for alleviating the global pain epidemic.

6.2.6 Creating a Deeper Specialized Metabolite Database

A significant resource for the field of plant specialized metabolism was the creation of publically accessible transcriptome libraries of over one thousand plant species (Xiao et al.,

2013; Matasci et al., 2014; Facchini et al., 2012). These initiatives have led to a wealth of

sequencing information that can be used for the discovery of novel biosynthetic genes using on

site or in house pattern matching algorithms. Conversely, our best metabolite databases contain

sparse information with regards to plant specialized metabolites. This represents a major

limitation to the gene discovery processes and limits our ability for understanding the physiology

of specialized metabolites at the systems level. As such, a future initiative should aim to

duplicate the transcriptome effort by building publically accessible specialized metabolite

database(s). With the rapid development of analytical platforms capable of turning out massive

metabolite data, our focus should now turn to annotating this data in a meaningful way. 157

Ultimately, the success of these initiatives will rely on collaborations with specialized metabolite

scientists, compound structural elucidation labs, and bioinformatics labs.

6.3 Conclusion

In conclusion, my PhD research has successfully expanded upon previous knowledge of

ODD-mediated O-dealkylation reactions in BIA metabolism in opium poppy, while at the same time outlining the discovery and characterization of an AKR/CYP fusion protein that is responsible for the stereochemical inversion of reticuline, thus completing all perceived biosynthetic steps in the pathway to morphine in opium poppy. These discoveries have significantly added to our understanding of BIA metabolism in opium poppy, and could have widereaching implications for synthetic biology applications, and for understanding the metabolism of other plant specialized metabolites. My thesis took advantage of (i) deep

transcript databases, (ii) homology-based cloning, (iii) reverse genetics, and (iv) state of the art

chromatography and mass spectrometry techniques to better understand the roles of ODDs in

BIA metabolism and to discover the enzymes responsible for the stereochemical inversion of

reticuline in opium poppy. My thesis has raised interesting new questions regarding BIA

metabolism. For example, do other BIA producing plant species contain ODDs involved in BIA

metabolism? Is ODD-mediated O-dealkylation more widespread in plant metabolism than

previously thought? What is the significance of the REPI fusion protein for the production of

morphinan alkaloids? Do other plant specialized metabolite pathways contain gene fusions? My

thesis outlines methodologies that are applicable to these and other questions, and can be used as

a guide for pursuing future investigations in plant secondary metabolism.

158

References

Akashi, T., Fukuchi-Mizutani, M., Aoki, T., Ueyama, Y., Yonekura-Sakakibara, K., Tanaka, Y., Kusumi, T. and Ayabe, S. (1999) Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones. Plant Cell Physiol., 40, 1182–1186.

Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A.C., Fist, A.J., Gerlach, W.L. and Larkin, P.J. (2004) RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol., 22, 1559–1566.

Allen, R.S., Miller, J.A., Chitty, J.A., Fist, A.J., Gerlach, W.L and Larkin, P.J. (2008) Metabolic engineering of morphinan alkaloids by over- expression and RNAi suppression of salutaridinol 7-O-acetyl-transferase in opium poppy. Plant Biotechnol J., 6, 22–30.

Amann, M. and H. Zenk, M. (1987) Preparation of dehydrobenzylisoquinolines by immobilized (S)-tetrahydroprotoberberine oxidase from plant cell cultures. Phytochemistry, 26, 3235–3240.

Amann, M., Nagakura, N. and Zenk, M.H. (1988) Purification and properties of (S) tetrahydroprotoberberine oxidase from suspension-cultured cells of Berberis wilsoniae. Eur. J. Biochem., 175, 17–25.

Bahadur, S. and Shukla, A.K. (1983) Studies on native medicinal plants, I. The quaternary alkaloids of Thalictrum javanicum. J. Nat. Prod., 46, 454–457.

Ballance, G.M. and Dixon, R. a (1995) Medicago sativa cDNAs encoding chalcone reductase. Plant Physiol., 107, 1027–1028.

Bartels, D., Engelhardt, K., Roncarati, R., Schneider, K., Rotter, M. and Salamini, F. (1991) An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein. EMBO J., 10, 1037–1043.

Bashir, K., Inoue, H., Nagasaka, S., Takahashi, M., Nakanishi, H., Mori, S. and Nishizawa, N.K. (2006) Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants. J. Biol. Chem., 281, 32395–32402.

Battersby, A.R., Foulkes, D.M. and Binks, R. (1965) Alkaloid biosynthesis. Part VIII. Use of optically active precursors for investigation on the biosynthesis of morphine alkaloids. J. Chem. Soc., 3323–3332.

Battersby, A.R., Foulkes, D.M., Hirst, M., Parry, G. V. and Staunton, J. (1968) Alkaloid biosynthesis. Part XI. Studies related to the formation and oxidation of reticuline in morphine biosynthesis. J. Chem. Soc. (C) Org., 210–216.

159

Beaudoin, G.A.W. and Facchini, P.J. (2013) Isolation and characterization of a cDNA encoding (S)-cis-N-methylstylopine 14-hydroxylase from opium poppy, a key enzyme in sanguinarine biosynthesis. Biochem. Biophys. Res. Commun., 431, 597–603.

Beaudoin, G.A.W. and Facchini, P.J. (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta, 240, 19–32.

Bella, A.J. and Brock, G.B. (2004) Intracavernous pharmacotherapy for erectile dysfunction. Endocrine, 23, 149–155.

Berim, A., Kim, M.-J. and Gang, D.R. (2014) Identification of a unique 2-oxoglutarate dependent flavone 7-O-demethylase completes the elucidation of the lipophilic flavone network in basil. Plant Cell Physiol., 56, 126–136.

Bernáth, J. (1998) Poppy: The Genus Papaver (Bernáth, J., Ed.), pp.1–2. Harwood Academic, Amsterdam, NL.

Bernhardt, R. (2006) Cytochromes P450 as versatile biocatalysts. J. Biotechnol., 124, 128–145.

Brochmann-Hanssen, E., Chen, C.H., Chen, C.R., Chiang, H.C., Leung, A.Y. and McMurtrey, K. (1975) Opium alkaloids. Part XVI. The biosynthesis of 1 benzylisoquinolines in Papaver somniferum. Preferred and secondary pathways; stereochemical aspects. J. Chem. Soc. Perkin 1, 1531–1537.

Brochmann-Hanssen, E., Leung, A.Y., Fu, C.C. and Zanati, G. (1971) Opium alkaloids. X. Biosynthesis of 1-benzylisoquinolines. J. Pharm. Sci., 60, 1672–1676.

Brodelius, M., Lundgren, A., Mercke, P. and Brodelius, P.E. (2002) Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum. Eur. J. Biochem., 269, 3570–3577.

Burch-Smith, T.M., Anderson, J.C., Martin, G.B. and Dinesh-Kumar, S.P. (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J., 39, 734–746.

Chauhan, S.P., Patra, N.K. and Srivastava, N.K. (1987) Dwarf mutant of Papaver somniferum with high morphine content. Mutat. Breed. Newsl., 30, 6.

Chenna, R. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res., 31, 3497–3500.

Cheong, Y.H., Chang, H.-S., Gupta, R., Wang, X., Zhu, T. and Luan, S. (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol., 129, 661–677.

160

Cho, J.-N., Ryu, J.-Y., Jeong, Y.-M., Park, J., Song, J.-J., Amasino, R.M., Noh, B. and Noh, Y.-S. (2012) Control of seed germination by light-induced histone arginine demethylation activity. Dev. Cell, 22, 736–748.

Choi, K.B., Morishige, T., Shitan, N., Yazaki, K. and Sato, F. (2002) Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem., 277, 830–835.

Clifton, I.J., McDonough, M.A., Ehrismann, D., Kershaw, N.J., Granatino, N. and Schofield, C.J. (2006) Structural studies on 2-oxoglutarate oxygenases and related double- stranded beta-helix fold proteins. J. Inorg. Biochem., 100, 644–669.

Collu, G., Unver, N., Peltenburg-Looman, A.M., Heijden, R. van der, Verpoorte, R. and Memelink, J. (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett., 508, 215–220.

Cortijo, J., Villagrasa, V., Pons, R., Berto, L., Martí-Cabrera, M., Martinez-Losa, M., Domenech, T., Beleta, J. and Morcillo, E.J. (1999) Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes. Br. J. Pharmacol., 127, 1641–1651.

Croteau, R., Ketchum, R.E.B., Long, R.M., Kaspera, R. and Mark, R. (2010) Taxol biosynthesis and molecular genetics. Phytochem. Rev., 5, 75–97.

Dang, T.T.T., Chen, X. and Facchini, P.J. (2014) Acetylation serves as a protective group in noscapine biosynthesis in opium poppy. Nat. Chem. Biol., 11, 104–106.

Dang, T.T.T. and Facchini, P.J. (2012) Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy. Plant Physiol. 159, 618–631.

Dang, T.T.T. and Facchini, P.J. (2014) Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy. FEBS Lett., 588, 198–204.

Dang, T.T.T. and Facchini, P.J. (2014) CYP82Y1 is N-methylcanadine 1-hydroxylase, a key noscapine biosynthetic enzyme in opium poppy. J. Biol. Chem., 289, 2013–2026.

Dang, T.T.T., Onoyovwi, A., Farrow, S.C., and Facchini, P.J. (2012) Biochemical genomics for gene discovery in benzylisoquinoline alkaloid biosynthesis in opium poppy and related species. Methods Enzymol. 515, 231–266

De Carolis, E. and De Luca, V. (1993) Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. J. Biol. Chem., 268, 5504–5511.

De Carolis, E. and De Luca, V. (1994) 2-Oxoglutarate-dependent dioxygenase and related enzymes: Biochemical characterization. Phytochemistry, 36, 1093–1107.

161

De Carolis, E., Chan, F., Balsevich, J. and Luca, V. De (1990) Isolation and characterization of a 2-oxoglutarate dependent dioxygenase involved in the second-to-last step in vindoline biosynthesis. Plant Physiol., 94, 1323–1329.

Decker, G., Wanner, G., Zenk, M.H. and Lottspeich, F. (2000) Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing. Electrophoresis, 21, 3500–3516.

De-Eknamkul, W. and Zenk, M.H. (1992) Purification and properties of 1,2-dehydroreticuline reductase from Papaver somniferum seedlings. Phytochemistry, 31, 813–821.

DeLoache, W.C., Russ, Z.N., Narcross, L., Gonzales, A.M., Martin, V.J.J. and Dueber, J.E. (2015) An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nat. Chem. Biol., 11, 465–471

Desgagné-Penix, I. and Facchini, P.J. (2012) Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. Plant J., 72, 331–344.

Desgagné-Penix, I., Farrow, S.C., Cram, D., Nowak, J. and Facchini, P.J. (2012) Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy. Plant Mol. Biol., 79, 295–313.

Dewick, P.M. (2009) Medicinal Natural Products, Chichester, UK: John Wiley & Sons, Ltd.

Díaz Chávez, M.L., Rolf, M., Gesell, A. and Kutchan, T.M. (2011) Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana. Arch. Biochem. Biophys., 507, 186–193.

Dinesh-Kumar, S.P.S., Anandalakshmi, R., Marathe, R., Schiff, M. and Liu, Y. (2003) Virus-induced gene silencing. Methods Mol. Bio., 236, 287–293.

Dittrich, H. and Kutchan, T.M. (1991) Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. Proc. Natl. Acad. Sci. U. S. A., 88, 9969–9973.

Dolejš, L. and Hanuš, V. (1967) Mass spectrometry of rhoeadine type alkaloids. Tetrahedron, 23, 2997–3005.

Durrens, P., Nikolski, M. and Sherman, D. (2008) Fusion and fission of genes define a metric between fungal genomes. PLOS Computational Biology, 4, e1000200.

Elleuche, S. (2015) Bringing functions together with fusion enzymes-from nature’s inventions to biotechnological applications. Appl. Microbial. Biotechnol. 99, 1545–1556.

162

Everard, J.D., Cantini, C., Grumet, R., Plummer, J. and Loescher, W.H. (1997) Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery. Plant Physiol., 113, 1427–1435.

Facchini, P.J. and De Luca, V. (1994) Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy. J. Biol. Chem., 269, 26684–26690.

Facchini, P.J. and Park, S.U. (2003) Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry, 64, 177–186.

Facchini, P.J. and St-Pierre, B. (2005) Synthesis and trafficking of alkaloid biosynthetic enzymes. Curr. Opin. Plant Biol., 8, 657–666.

Facchini, P.J., Bohlmann, J., Covello, P.S., et al. (2012) Synthetic biosystems for the production of high-value plant metabolites. Trends Biotechnol., 30, 127–131.

Farrow, S.C. and Facchini, P.J. (2013) Dioxygenases catalyze O-demethylation and O,O demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy. J. Biol. Chem., 288, 1–5.

Farrow, S.C. and Facchini, P.J. (2014) Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism. Front. Plant Sci., 5, 1–15.

Farrow, S.C., Hagel, J.M. and Facchini, P.J. (2012) Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids. Phytochemistry, 77, 79–88.

Farrow, S.C., Hagel, J.M., Beaudoin, G. a W., Burns, D.C. and Facchini, P.J. (2015) Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Nat. Chem. Biol. 11, 728–723.

Ferndahl, C., Bonander, N., Logez, C., Wagner, R., Gustafsson, L., Larsson, C., Hedfalk, K., Darby, R. a J. and Bill, R.M. (2010) Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory. Microb. Cell Fact., 9, 47.

Field, B. and Osbourn, A.E. (2008) Metabolic diversification-independent assembly of operon- like gene clusters in different plants. Science, 320, 543–547.

Flashman, E. and Schofield, C.J. (2007) The most versatile of all reactive intermediates? Nat. Chem. Biol., 3, 86–87.

Frey, M., Huber, K., Park, W.J., Sicker, D., Lindberg, P., Meeley, R.B., Simmons, C.R., Yalpani, N. and Gierl, A. (2003) A 2-oxoglutarate-dependent dioxygenase is integrated in DIMBOA-biosynthesis. Phytochemistry, 62, 371–376.

163

Frick, S., Kramell, R., Schmidt, J., Fist, A.J. and Kutchan, T.M. (2005) Comparative qualitative and quantitative determination of alkaloids in narcotic and condiment Papaver somniferum cultivars. J. Nat. Prod., 68, 666–673.

Frick, S., Kramell, R. and Kutchan, T.M. (2007) Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding. Metab. Eng. 9, 169–176.

Fujii, N., Inui, T., Iwasa, K., Morishige, T. and Sato, F. (2007) Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway incultured California poppy cells. TransgenicRes., 16, 363–375.

Fukuto, J.M., Kumagai, Y. and Cho, A.K. (1991) Determination of the mechanism of demethylenation of (methylenedioxy)phenyl compounds by cytochrome P450 using deuterium isotope effects. J. Med. Chem., 34, 2871–2876.

Gavidia, I., Pérez-Bermúdez, P. and Seitz, H.U. (2002) Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves. Eur. J. Biochem., 269, 2842–2850.

Gerardy, R. and Zenk, M.H. (1992) Formation of salutaridine from (R)-reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum. Phytochemistry, 32, 79–86.

Gesell, A., Rolf, M., Ziegler, J., Chávez, M.L.D., Huang, F.C. and Kutchan, T.M. (2009) CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J. Biol. Chem., 284, 24432–24442.

Gorres, K.L. and Raines, R.T. (2010) Prolyl 4-hydroxylase. Crit. Rev. Biochem. Mol. Biol., 45, 106–124.

Gould, B. and Kramer, E.M. (2007) Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae). Plant Methods, 3, 6.

Grobe, N., Zhang, B., Fisinger, U., Kutchan, T.M., Zenk, M.H. and Guengerich, F.P. (2009) Mammalian cytochrome P450 enzymes catalyze the phenol-coupling step in endogenous morphine biosynthesis. J. Biol. Chem., 284, 24425–24431.

Grothe, T., Lenz, R. and Kutchan, T.M. (2001) Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum. J. Biol. Chem., 276, 30717–30723.

Groves, J.T. (2006) High-valent iron in chemical and biological oxidations. J. Inorg. Biochem., 100, 434–447.

Guengerich, F.P. (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol., 14, 611–650.

164

Hagel, J.M. and Facchini, P.J. (2010a) Biochemistry and occurrence of O-demethylation in plant metabolism. Front. Physiol., 1, 14.

Hagel, J.M. and Facchini, P.J. (2010b) Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat. Chem. Biol., 6, 273–275.

Hagel, J.M. and Facchini, P.J. (2013) Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 4, 647-672

Hagel, J.M., Beaudoin, G.A.W., Fossati, E., Ekins, A., Martin, V.J.J. and Facchini, P.J. (2012) Characterization of a flavoprotein oxidase from opium poppy catalyzing the final steps in sanguinarine and papaverine biosynthesis. J. Biol. Chem., 287, 42972–42983.

Hagel, J.M., Morris, J.S., Lee, E.-J., et al. (2015) Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants. BMC Plant Biol., 15, 227.

Halkier, B.A. and Du, L. (1997) The biosynthesis of glucosinolates. Trends Plant Sci., 2, 425– 431.

Han, X., Lamshöft, M., Grobe, N., Ren, X., Fist, A.J., Kutchan, T.M., Spiteller, M. and Zenk, M.H. (2010) The biosynthesis of papaverine proceeds via (S)-reticuline. Phytochemistry, 71, 1305–1312.

Hangasky, J.A., Taabazuing, C.Y., Valliere, M.A. and Knapp, M.J. (2013) Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the α-KG dependent oxygenases. Metallomics, 5, 287–301.

Hannah, M.A., Heyer, A.G. and Hincha, D.K. (2005) A global survey of gene regulation during cold acclimation in Arabidopsis thaliana. PLoS Genet., 1, 179–196.

Hannon, G.J. (2002) RNA interference. Nature, 418, 244–251.

Hansen, B.G., Kerwin, R.E., Ober, J.A., Lambrix, V.M., Mitchell-Olds, T., Gershenzon, J., Halkier, B.A. and Kliebenstein, D.J. (2008) A novel 2-oxoacid-dependent dioxygenase involved in the formation of the goiterogenic 2-hydroxybut-3-enyl glucosinolate and generalist insect resistance in Arabidopsis,. Plant Physiol., 148, 2096–2108.

Hanson, A.D., Pribat, A., Waller, J.C., de Crecy-Iagard, V. (2011) ‘Unknown’ proteins and ‘orphan’ enzymes:the missing half of the engineering parts list – and how to find it. Biochem. J., 425, 1-11.

Hashimoto, T. and Yamada, Y. (1986) Hyoscyamine 6-beta-hydroxylase, a 2-oxoglutarate dependent dioxygenase, in alkaloid-producing root cultures. Plant Physiol., 81, 619–625.

165

Hashimoto, T. and Yamada, Y. (1987) Purification and characterization of hyoscyamine 6 beta-hydroxylase from root cultures of Hyoscyamus niger L. hydroxylase and epoxidase activities in the enzyme preparation. Eur. J. Biochem., 164, 277–285.

Hayaishi, O. and Hashimoto, K. (1950) Pyrocatecase a new enzyme catalyzing oxidative breakdown of pyrocatechin. J. Biochem., 37, 371–374.

Hayaishi, O., Shimazono, H., Katagiri, M. and Saito, Y. (1956) Enzymatic formation of oxalate and acetate from oxaloacetate. J. Am. Chem. Soc., 78, 5126–5127.

He, X. and Brossi, A. (1993) 1,2-Dehydroreticuline: Conversion of iminium salts into enamines. J. Nat. Prod., 56, 973–975.

Henricsson, C., Jesus Ferreira, M.C. de, Hedfalk, K., Elbing, K., Larsson, C., Bill, R.M., Norbeck, J., Hohmann, S. and Gustafsson, L. (2005) Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations. Appl. Environ. Microbiol., 71, 6185–6192.

Hileman, L.C., Drea, S., Martino, G. De, Litt, A. and Irish, V.F. (2005) Virus-induced gene silencing is an effective tool for assaying gene function in the basal eudicot species Papaver somniferum (opium poppy). Plant J., 44, 334–341.

Hirata, K., Poeaknapo, C., Schmidt, J. and Zenk, M.H. (2004) 1,2-Dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry, 65, 1039–1046.

Hutton, J.J., Tappel, A.L. and Udenfriend, S. (1967) Cofactor and substrate requirements of collagen proline hydroxylase. Arch. Biochem. Biophys., 118, 231–240.

Ibragimova, M.U., Israilov, I.A., Yunusov, M.S. and Yunusov, S.Y. (1974) Alkaloids of Fumaria vaillantii structure of vaillantine. Chem. Nat. Compd., 10, 481–482.

Ikezawa, N., Iwasa, K. and Sato, F. (2007) Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica. FEBS J., 274, 1019–1035.

Ikezawa, N., Iwasa, K. and Sato, F. (2008) Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S) reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells. J. Biol. Chem., 283, 8810–8821.

Ikezawa, N., Iwasa, K. and Sato, F. (2009) CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica. Plant Cell Rep., 28, 123–133.

166

Ikezawa, N., Tanaka, M., Nagayoshi, M., Shinkyo, R., Sakaki, T., Inouye, K. and Sato, F. (2003) Molecular cloning and characterization of CYP719, a methylenedioxy bridge- forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells. J. Biol. Chem., 278, 38557–38565.

Im, S.C. and Waskell, L. (2011) The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b5. Arch. Biochem. Biophys., 507, 144–153.

Itkin, M., Heinig, U., Tzfadia, O., et al. (2013) Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science, 341, 175–179.

Jin, C.W., You, G.Y., He, Y.F., Tang, C., Wu, P. and Zheng, S.J. (2007) Iron deficiency- induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover. Plant Physiol., 144, 278–285.

Jirschitzka, J., Schmidt, G.W., Reichelt, M., Schneider, B., Gershenzon, J. and D’Auria, J.C. (2012) Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae. Proc. Natl. Acad. Sci. U. S. A., 109, 10304–10309.

Jonczyk, R., Schmidt, H., Osterrieder, A., et al. (2008) Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7. Plant Physiol., 146, 1053–1063.

Kai, K., Mizutani, M., Kawamura, N., Yamamoto, R., Tamai, M., Yamaguchi, H., Sakata, K. and Shimizu, B. (2008) Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana. Plant J., 55, 989– 999.

Kametani, T. (1968) The Chemistry of the Isoquinoline Alkaloids. Hirokawa: Tokyo, and Elsevier: Amsterdam

Kametani, T. (1974) The Chemistry of the Isoquinoline Alkaloids, Vol. 2. The Sendai Institute of Heterocyclic Chemistry: Japan

Kanayama, Y., Mori, H., Imaseki, H. and Yamaki, S. (1992) Nucleotide Sequence of a cDNA Encoding NADP-Sorbitol-6-Phosphate Dehydrogenase from Apple. Plant Physiol., 100, 1607–1608.

Kavšček, M., Stražar, M., Curk, T., Natter, K. and Petrovič, U. (2015) Yeast as a cell factory: current state and perspectives. Microb. Cell Fact., 14, 94.

Kawai, Y., Ono, E. and Mizutani, M. (2014) Evolution and diversity of the 2-oxoglutarate dependent dioxygenase superfamily in plants. Plant J., 78, 328–343.

167

Kempe, K., Higashi, Y., Frick, S., Sabarna, K., Kutchan, T.M. (2009) RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes. Phytochemistry, 70, 579–589.

Kirby, G.W. (1967) Biosynthesis of the morphine alkaloids. Science, 155, 170–173.

Kliebenstein, D.J., Lambrix, V.M., Reichelt, M., Gershenzon, J. and Mitchell-Olds, T. (2001) Gene duplication in the diversification of secondary metabolism: tandem 2 oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis. Plant Cell, 13, 681–693. Kraus, P.F. and Kutchan, T.M. (1995) Molecular cloning and heterologous expression of a cDNA encoding berbamunine synthase, a C-O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Proc. Natl. Acad. Sci. U. S. A., 92, 2071–2075.

Lamb, D.C., Lei, L., Warrilow, A.G.S., Lepesheva, G.I., Mullins, J.G.L., Waterman, M.R. and Kelly, S.L. (2009) The first virally encoded cytochrome p450. J. Virol., 83, 8266–8269.

Lan, P., Li, W., Wen, T.-N., Shiau, J.-Y., Wu, Y.-C., Lin, W. and Schmidt, W. (2011) iTRAQ protein profile analysis of Arabidopsis roots reveals new aspects critical for iron homeostasis. Plant Physiol., 155, 821–834.

Larkin, M.A., Blackshields, G., Brown, N.P., et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948.

Lasskaya, O.E. and Tolkachev, O.N. (1978) Quaternary benzophenanthridine alkaloids 9,10 demethylene derivatives of sanguinarine. Chem. Nat. Compd., 14, 650–652.

Lee, E.-J. and Facchini, P. (2010) Norcoclaurine synthase is a member of the pathogenesis- related 10/Bet v1 protein family. Plant Cell, 22, 3489–3503.

Lee, E.-J. and Facchini, P.J. (2011) Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy. Plant Physiol., 157, 1067–1078.

Lin, L.Y., Kumagai, Y. and Cho, A.K. (1992) Enzymic and chemical demethylenation of (methylenedioxy)amphetamine and (methylenedioxy)methamphetamine by rat brain microsomes. Chem. Res. Toxicol., 5, 401–406.

Lindahl, T., Sedgwick, B., Sekiguchi, M. and Nakabeppu, Y. (1988) Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem., 57, 133–157.

Liscombe, D.K. and Facchini, P.J. (2007) Molecular cloning and characterization of tetrahydroprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis in opium poppy. J. Biol. Chem., 282, 14741–14751.

Liu, C. and Dixon, R.A. (2001) Elicitor-induced association of isoflavone. Society, 13, 2643– 2658.

168

Liu, J.K. and Couldwell, W.T. (2005) Intra-arterial papaverine infusions for the treatment of cerebral vasospasm induced by aneurysmal subarachnoid hemorrhage. Neurocrit. Care, 2, 124–132.

Liu, Y., Hao, H., Xie, H., Hua, L.V., Liu, C. and Wang, G. (2009) Oxidative demethylenation and subsequent glucuronidation are the major metabolic pathways of berberine in rats. J. Pharm. Sci., 98, 4391–4401.

Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real- -ΔΔC time quantitative PCR and the 2 T method. Methods, 25, 402–408.

Loenarz, C. and Schofield, C.J. (2008) Expanding chemical biology of 2-oxoglutarate oxygenases. Nat. Chem. Biol., 4, 152–156.

Loescher, W.H., Tyson, R.H., Everard, J.D., Redgwell, R.J. and Bieleski, R.L. (1992) Mannitol synthesis in higher plants 1. Plant Physiol., 98, 1396–1402.

Manoj, K.M., Baburaj, A., Ephraim, B., et al. (2010) Explaining the atypical reaction profiles of heme enzymes with a novel mechanistic hypothesis and kinetic treatment. PLoS One, 5.

Marbaix, A.Y., Noël, G., Detroux, A.M., Vertommen, D., Schaftingen, E. Van and Linster, C.L. (2011) Extremely conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide. J. Biol. Chem., 286, 41246–41252.

Marienhagen, J. and Bott, M. (2013) Metabolic engineering of microorganisms for the synthesis of plant natural products. J. Biotechnol., 163, 166–178.

Martens, S. and Mithöfer, A. (2005) Flavones and flavone synthases. Phytochemistry, 66, 2399–2407.

Martens, S., Preuss, A. and Matern, U. (2010) Multifunctional flavonoid dioxygenases: flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L. Phytochemistry, 71, 1040–1049.

Matasci, N., Hung, L.-H., Yan, Z., et al. (2014) Data access for the 1,000 Plants (1KP) project. Gigascience, 3, 17.

Matsumoto, S., Mizutani, M., Sakata, K. and Shimizu, B.-I. (2012) Molecular cloning and functional analysis of the ortho-hydroxylases of p-coumaroyl coenzyme A/feruloyl coenzyme A involved in formation of umbelliferone and scopoletin in sweet potato, Ipomoea batatas (L.) Lam. Phytochemistry, 74, 49–57.

Meisel, L., Fonseca, B., GonzÁlez, S., et al. (2005) A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biol. Res., 38, 83–88.

169

Meunier, B., Visser, S.P. de and Shaik, S. (2004) Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes. Chem. Rev., 104, 3947–3980.

Meyer, M.R., Peters, F.T. and Maurer, H.H. (2008) The role of human hepatic cytochrome P450 isozymes in the metabolism of racemic 3,4-methylenedioxymethamphetamine and its enantiomers. Pharmacology, 36, 2345–2354.

Meyer, M.R., Peters, F.T. and Maurer, H.H. (2009) Investigations on the human hepatic cytochrome P450 isozymes involved in the metabolism of 3,4-methylenedioxy amphetamine (MDA) and benzodioxolyl-butanamine (BDB) enantiomers. Toxicol. Lett., 190, 54–60.

Mielecki, D., Zugaj, D.Ł., Muszewska, A., Piwowarski, J., Chojnacka, A., Mielecki, M., Nieminuszczy, J., Grynberg, M. and Grzesiuk, E. (2012) Novel AlkB dioxygenases alternative models for in silico and in vivo studies. PLoS One, 7, e30588.

Millan, M.J., Maiofiss, L., Cussac, D., Audinot, V., Boutin, J.-A. and Newman-Tancredi, A. (2002) Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. I. A multivariate analysis of the binding profiles of 14 drugs at 21 native and cloned human receptor subtypes. J. Pharmacol. Exp. Ther., 303, 791–804.

Minami, H., Kim, J.-S., Ikezawa, N., Takemura, T., Katayama, T., Kumagai, H. and Sato, F. (2008) Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. U. S. A., 105, 7393–7398.

Mizutani, M. and Ohta, D. (2010) Diversification of P450 genes during land plant evolution. Annu. Rev. Plant Biol., 61, 291–315.

Mizutani, M. and Sato, F. (2011) Unusual P450 reactions in plant secondary metabolism. Arch. Biochem. Biophys., 507, 194–203.

Montgomery, C.T., Cassels, B.K. and Shamma, M. (1983) The rhoeadine alkaloids. J. Nat. Prod., 46, 441–453.

Morikawa, T., Mizutani, M., Aoki, N., et al. (2006) Cytochrome P450 CYP710A encodes the sterol C-22 desaturase in Arabidopsis and tomato. Plant Cell, 18, 1008–1022.

Morishige, T., Dubouzet, E., Choi, K.B., Yazaki, K. and Sato, F. (2002) Molecular cloning of columbamine O-methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem., 269, 5659–5667.

Morishige, T., Tsujita, T., Yamada, Y. and Sato, F. (2000) Molecular characterization of the S-adenosyl-L-methionine:3’-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid biosynthesisin Coptis japonica. J. Biol. Chem., 275, 23398–23405.

170

Morrison, J.P., Read, J.A., Coleman, W.G. and Tanner, M.E. (2005) Dismutase activity of ADP-L-glycero-D-manno-heptose 6-epimerase: Evidence for a direct oxidation/reduction mechanism. Biochemistry, 44, 5907–5915.

Müller-Schweinitzer, E., Hasse, J. and Swoboda, L. (1993) Cryopreservation of human bronchi. J. Asthma, 30, 451–457.

Nakajima, N., Mori, H., Yamazaki, K. and Imaseki, H. (1990) Molecular Cloning and Sequence of a Complementary DNA encoding 1-aminocyclopropane-l-carboxylate synthase induced by tissue wounding. Plant Cell Physiol., 31, 1021–1029.

Nakanishi, H., Okumura, N., Umehara, Y., Nishizawa, N.K., Chino, M. and Mori, S. (1993) Expression of a gene specific for iron deficiency (Ids3) in the roots of Hordeum vulgare. Plant Cell Physiol., 34, 401–10.

Nakanishi, H., Yamaguchi, H., Sasakuma, T., Nishizawa, N.K. and Mori, S. (2000) Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores. Plant Mol. Biol., 44, 199–207.

Nelson, D. and Werck-Reichhart, D. (2011) A P450-centric view of plant evolution. Plant J., 66, 194–211.

Nützmann, H.W. and Osbourn, A. (2014) Gene clustering in plant specialized metabolism. Curr. Opin. Biotechnol., 26, 91–99.

Oberschall, A., Deák, M., Török, K., Sass, L., Vass, I., Kovács, I., Fehér, A., Dudits, D. and Horváth, G. V. (2000) A novel aldose/aldehyde reductase protects transgenic plants against lipid peroxidation under chemical and drought stresses. Plant J., 24, 437–446.

Okumura, N., Nishizawa, N., Umehara, Y., Ohata, T., Nakanishi, H., Yamaguchi, T., Chino, M. and Mori, S. (1994) A dioxygenase gene (Ids2) expressed under iron deficiency conditions in the roots of Hordeum vulgare. Plant Mol. Biol., 25, 705–719.

Olsen, J.G., Pedersen, L., Christensen, C.L., Olsen, O. and Henriksen, A. (2008) Barley aldose reductase: structure, cofactor binding, and substrate recognition in the aldo/keto reductase 4C family. Proteins Struct. Funct. Genet., 71, 1572–1581.

Pathak, S., Lakhwani, D., Gupta, P., Mishra, B.K., Shukla, S., Asif, M.H. and Trivedi, P.K. (2013) Comparative transcriptome analysis using high papaverine mutant of Papaver somniferum reveals pathway and uncharacterized steps of papaverine biosynthesis. PLoS One, 8.

Paul, L.D., Springer, D., Staack, R.F., Kraemer, T. and Maurer, H.H. (2004) Cytochrome P450 isoenzymes involved in rat liver microsomal metabolism of californine and protopine. Eur. J. Pharmacol., 485, 69–79.

171

Pauli, H.H. and Kutchan, T.M. (1998) Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3’-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J., 13, 793–801.

Penning, T.M. (2014) The aldo-keto reductases (AKRs): Overview. Chem. Biol. Interact., 234, 236–246.

Peters, R.J., Carter, O. a., Zhang, Y., Matthews, B.W. and Croteau, R.B. (2003) Bifunctional abietadiene synthase: Mutual structural dependence of the active sites for protonation-initiated and ionization-initiated cyclizations. Biochemistry, 42, 2700–2707.

Pictet, A., and Spengler, T. (1911). Uber die bildung von isochinolin-derivaten durch einwirkung von methylal auf phenyl-athylamin, phenyl-alanin und tyrosine. Ber. Dtsch. Chem. Ges. 44, 2030–2036.

Pillai, B., Cherney, M.M., Diaper, C.M., Sutherland, A., Blanchard, J.S., Vederas, J.C. and James, M.N.G. (2006) Structural insights into stereochemical inversion by diaminopimelate epimerase: an antibacterial drug target. Proc. Natl. Acad. Sci. U. S. A., 103, 8668–8673.

Poulton, J.E. (1981) Transmethylation and demethylation reaction in the metabolism of secondary plant products. In: Stumpf, P.K., and Conn, E.E., (eds.) The Biochemistry of Plants, 7, 668–723.

Prescott, A.G. and John, P. (1996) Dioxygenases: molecular structure and role in plant metabolism. Annu. Rev. Plant Physiol. Plant Mol. Biol., 47, 245–271.

Purpero, V. and Moran, G.R. (2007) The diverse and pervasive chemistries of the α-keto acid dependent enzymes. J. Biol. Inorg. Chem., 12, 587–601.

Quasdorf, K.W. and Overman, L.E. (2014) Catalytic enantioselective synthesis of quaternary carbon stereocentres. Nature, 516, 181–191.

Rathbone, D.A. and Bruce, N.C. (2002) Microbial transformation of alkaloids. Curr. Opin. Microbiol., 5, 274–281.

Rižner, T.L. and Penning, T.M. (2014) Role of aldo-keto reductase family 1 (AKR1) enzymes in human steroid metabolism. Steroids, 79, 49–63.

Ro, D-K., Paradise, E.M., Ouellet, M., et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature, 440, 940–943.

Robinson, R. (1917). A theory of the mechanism of the phytochemical synthesis of certain alkaloids. J. Chem. Soc. Trans. 111, 876–899.

172

Rönsch, H. (1977) Biosynthesis of alpinigenine by way of tetrahydroprotoberberine and protopine intermediates. Phytochemistry, 16, 691–698.

Rueffer, M. and Zenk, M.H. (1987) Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans. Tetrahedron Lett., 28, 5307–5310.

Rueffer, M., El-Shagi, H., Nagakura, N. and Zenk, M.H. (1981) (S)-norlaudanosoline synthase: the first enzyme in the benzylisoquinoline biosynthetic pathway. FEBS Lett., 129, 5–9. Ruiz, M.T., Voinnet, O. and Baulcombe, D.C. (1998) Initiation and maintenance of virus- induced gene silencing. Plant Cell, 10, 937–946.

Runguphan, W., Glenn, W.S. and O’Connor, S.E. (2012) Redesign of a dioxygenase in morphine biosynthesis. Chem. Biol., 19, 674–678.

Sallaud, C., el-Turk, J., Bigarré, L., Sevin, H., Welle, R. and Esnault, R. (1995) Nucleotide sequences of three chalcone reductase genes from alfalfa. Plant Physiol., 108, 869–870.

Samanani, N., Liscombe, D.K. and Facchini, P.J. (2004) Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J., 40, 302–313.

Schäfer, H. and Wink, M. (2009) Medicinally important secondary metabolites in recombinant microorganisms or plants: progress in alkaloid biosynthesis. Biotechnol. J., 4, 1684–1703.

Schiff, P.L. (1991) Bisbenzylisoquinoline Alkaloids. J. Nat. Prod., 54, 645–749.

Schmidt, J., Boettcher, C., Kuhnt, C., Kutchan, T.M. and Zenk, M.H. (2007) Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass 13 spectrometry after [ring- C6]-tyramine feeding. Phytochemistry, 68, 189–202.

Schmidt, J., Raith, K., Boettcher, C. and Zenk, M.H. (2005) Analysis of benzylisoquinoline type alkaloids by electrospray tandem mass spectrometry and atmospheric pressure photoionization. Eur. J. Mass Spectrom. (Chichester, Eng)., 11, 325–333.

Sengupta, D., Naik, D. and Reddy, A.R. (2015) Plant aldo-keto reductases (AKRs) as multi tasking soldiers involved in diverse plant metabolic processes and stress defense: a structure-function update. J. Plant Physiol., 179, 40–55.

Seya, M.J., Gelders, S.F., Achara, O.U., Milani, B., Scholten, W.K. (2011) A first comparison between the consumption of and the need for opioid analgesics at country, regional, and global levels. J. Pain Palliat. Care Pharmacother. 25, 6–18.

173

Sharma, H., Landau, M.J., Vargo, M.A., Spasov, K.A. and Anderson, K.S. (2013) First three-dimensional structure of toxoplasma gondii thymidylate synthase-dihydrofolate reductase: Insights for catalysis, interdomain interactions, and substrate channeling. Biochemistry, 52, 7305–7317.

Sillanpää, M. and Koponen, M. (1978) Papaverine in the prophylaxis of migraine and other vascular headache in children. Acta Paediatr. 67, 209-212.

Simpson, P.J., Tantitadapitak, C., Reed, A.M., Mather, O.C., Bunce, C.M., White, S.A. and Ride, J.P. (2009) Characterization of two novel aldo-keto reductases from Arabidopsis: expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress. J. Mol. Biol., 392, 465–480.

Stadler, R. and Zenk, M.H. (1990) A revision of the generally accepted pathway for the biosynthesis of the benzyltetrahydroisoquinoline alkaloid reticuline. Liebigs Ann. der Chemie, 1990, 555–562.

Stadler, R. and Zenk, M.H. (1993) The purification and characterization of a unique cytochrome P-450 enzyme from Berberis stolonifera plant cell cultures. J. Biol. Chem., 268, 823–831.

Stadler, R., Kutchan, T., Loeffler, S., Nagakura, N., Cassels, B. and Zenk, M. (1987) Revision of the early steps of reticuline biosynthesis. Tetrahedron Lett., 28, 1251–1254.

Stadler, R., Kutchan, T.M. and Zenk, M.H. (1989) (S)-norcoclaurine is the central intermediate in benzylisoquinoline alkaloid biosynthesis. Phytochemistry, 28, 1083–1086.

Stapon, A., Li, R. and Townsend, C.A. (2003) Synthesis of (3S,5R)-Carbapenam-3-carboxylic acid and its role in cabapenem biosynthesis and the stereoinversion problem. J. Am. Chem. Soc., 125, 15746–15747.

Stermitz, F.R., Lorenz, P., Tawara, J.N., Zenewicz, L.A. and Lewis, K. (2000) Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5’-methoxyhydnocarpin, a multidrug pump inhibitor. Proc. Natl. Acad. Sci. U. S. A., 97, 1433–1437.

Stilio, V.S. di, Kumar, R.A., Oddone, A.M., Tolkin, T.R., Salles, P. and McCarty, K. (2010) Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum. PLoS One, 5.

Straeten, D. Van der, Wiemeersch, L. Van, Goodman, H.M. and Montagu, M. Van (1990) Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1 carboxylate synthase in tomato. Proc. Natl. Acad. Sci. U. S. A., 87, 4859–4863.

Takemura, T., Ikezawa, N., Iwasa, K. and Sato, F. (2013) Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells. Phytochemistry, 91, 100–108.

174

Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, J.H., Yamada, Y. and Sato, F. (1995) Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O methyltransferase from cultured cells of Coptis japonica. Plant Cell Physiol., 36, 29–36.

Takeuchi, K., Sakamoto, S., Nagayoshi, Y., Nishizawa, H. and Matsubara, J. (2004) Reactivity of the human internal thoracic artery to vasodilators in coronary artery bypass grafting. Eur. J. Cardiothorac. Surg., 26, 956–959.

Tanahashi, T. and Zenk, M.H. (1990) Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis. Phytochemistry, 29, 1113–1122.

Tang, H., Vasconcelos, A.C. and Berkowitz, G.A. (1995) Evidence that plant K+ channel proteins have two different types of subunits. Plant Physiol., 109, 327–330.

Tani, C. and Tagahara, K. (1977) Studies on the alkaloids of papaveraceous plants. XXVIII. The Biosynthesis of rhoeadine. J. Pharm. Soc. Japan, 97, 93–102.

Tarhonskaya, H., Szöllössi, A., Leung, I.K.H., et al. (2014) Studies on deacetoxycephalosporin C synthase support a consensus mechanism for 2-oxoglutarate dependent oxygenases. Biochemistry, 53, 2483–2493.

Thodey, K., Galanie, S. and Smolke, C.D. (2014) A microbial biomanufacturing platform for natural and semisynthetic opioids. Nat. Chem. Biol., 10, 1–10.

Thompson, M.A. (1980). Muscle relaxant drugs. Br. J. Hosp. Med., 23, 153–154.

Tiainen, P., Myllyharju, J. and Koivunen, P. (2005) Characterization of a second Arabidopsis thaliana prolyl 4-hydroxylase with distinct substrate specificity. J. Biol. Chem., 280, 1142– 1148.

Tracy, T.S. (2006) Atypical cytochrome P450 kinetics: implications for drug discovery. Drugs R&D. 7, 349–363

Trenchard, I.J. and Smolke, C.D. (2015) Engineering strategies for the fermentative production of plant alkaloids in yeast. Metab. Eng., 30, 96–104.

Tucker, G.T., Lennard, M.S., Ellis, S.W., Woods, H.F., Cho, A.K., Lin, L.Y., Hiratsuka, A., Schmitz, D.A. and Chu, T.Y.Y. (1994) The demethylenation of methylenedioxymethamphetamine (“ecstasy”) by debrisoquine hydroxylase (CYP2D6). Biochem. Pharmacol., 47, 1151–1156.

Unterlinner, B., Lenz, R. and Kutchan, T.M. (1999) Molecular cloning and functional expression of codeinone reductase: The penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J., 18, 465–475.

175

Van der Fits, L. van der and Memelink, J. (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289, 295–297.

Vazquez-Flota, F., Carolis, E. De, Alarco, A.M. and Luca, V. De (1997) Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don. Plant Mol. Biol., 34, 935–948.

Vialart, G., Hehn, A., Olry, A., et al. (2012) A 2-oxoglutarate-dependent dioxygenase from Ruta graveolens L. exhibits p-coumaroyl CoA 2’-hydroxylase activity (C2'H): a missing step in the synthesis of umbelliferone in plants. Plant J., 70, 460–470.

Vigani, G., Morandini, P. and Murgia, I. (2013) Searching iron sensors in plants by exploring the link among 2’-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency. Front. Plant Sci., 4, 169.

Wang, Y., Yi, H., Wang, M., Yu, O. and Jez, J.M. (2011) Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase. J. Am. Chem. Soc., 133, 20684–20687.

Wege, S., Scholz, A., Gleissberg, S. and Becker, A. (2007) Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): An evaluation of VIGS as a strategy to obtain functional data from non-model plants. Ann. Bot., 100, 641–649.

Welle, R., Schröder, G., Schiltz, E., Grisebach, H. and Schröder, J. (1991) Induced plant responses to pathogen attack. Eur. J. Biochem., 196, 423–430.

Werck-Reichhart, D. and Feyereisen, R. (2000) Cytochromes P450: a success story. Genome Biol., 1, 1–9 .

Werck-Reichhart, D., Bak, S. and Paquette, S. (2002) Cytochromes P450. The Arabidopsius Book., 2, 1–28.

Wickens, J.R., Sleeman, R. and Keely, B.J. (2006) Atmospheric pressure ionisation mass spectrometric fragmentation pathways of noscapine and papaverine revealed by multistage mass spectrometry and in-source deuterium labelling. Rapid Commun. Mass Spectrom., 20, 473–480.

Wijekoon, C.P. and Facchini, P.J. (2012) Systematic knockdown of morphine pathway enzymes in opium poppy using virus-induced gene silencing. Plant J., 69, 1052–1063.

Wilmouth, R.C., Turnbull, J.J., Welford, R.W.D., Clifton, I.J., Prescott, A.G. and Schofield, C.J. (2002) Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. Structure, 10, 93–103.

176

Wink, M. (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry, 64, 3–19.

Winterstein, E. and Trier, G. (1910) Die Alkaloide: Eine Monographie der Naturlichen Basen, pp. 307. Borntrager, Berlin, DE.

Winzer, T., Gazda, V., He, Z., et al. (2012) A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science, 336, 1704–1708.

Winzer, T., Kern, M., King, A.J., Larson, T.R., Teodor, R.I., Donninger, S.L., Li, Y., Dowle, A.A., Cartwright, J., Bates, R., Ashford, D., Thomas, J., Walker, C., Bowser, T.A. and Graham, I.A. (2015) Morphinan biosynthesis in opium poppy requires a P450 oxidoreductase fusion protein. Science, 349, 309–312.

Xiao, M., Zhang, Y., Chen, X., et al. (2013) Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest. J. Biotechnol., 166, 122–134.

Yamaguchi, S. (2008) Gibberellin metabolism and its regulation. Annu. Rev. Plant Biol., 59, 225–251.

Zenk, M.H., Gerardy, R. and Stadler, R. (1989) Phenol oxidative coupling of benzylisoquinoline alkaloids is catalysed by regio- and stereo-selective cytochrome P-450 linked plant enzymes: salutaridine and berbamunine. J. Chem. Soc. Chem. Commun., 1725.

Zhang, K., Halitschke, R., Yin, C., Liu, C.-J. and Gan, S.-S. (2013) Salicylic acid 3 hydroxylase regulates Arabidopsis leaf longevity by mediating salicylic acid catabolism. Proc. Natl. Acad. Sci. U. S. A., 110, 14807–14812.

Zhang, Z., Ren, J.-S., Clifton, I.J. and Schofield, C.J. (2004) Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase--the ethylene-forming enzyme. Chem. Biol., 11, 1383–1394.

Zhao, Z., Zhang, Y., Liu, X., et al. (2013) A role for a dioxygenase in auxin metabolism and reproductive development in rice. Dev. Cell, 27, 113–122.

Zhu, W. (2008) CYP2D6: a key enzyme in morphine synthesis in animals. Med. Sci. Monit., 14, SC15–C18.

Ziegler, J. and Facchini, P.J. (2008) Alkaloid biosynthesis: metabolism and trafficking. Annu. Rev. Plant Biol., 59, 735–69.

Ziegler, J., Brandt, W., Geissler, R. and Facchini, P.J. (2009) Removal of substrate inhibition and increase in maximal velocity in the short chain dehydrogenase/reductase salutaridine reductase involved in morphine biosynthesis. J. Biol. Chem., 284, 26758–67.

177

Ziegler, J., Voigtländer, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, C., Gesell, A. and Kutchan, T.M. (2006) Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J., 48, 177–192.

Zulak, K.G., Cornish, A., Daskalchuk, T.E., et al. (2007) Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism. Planta, 225, 1085–1106.

178

Appendix A.1. Chromatograhpic and mass spectral data used for the identification and relative quantification of benzylisoquinoline alkaloid enzymatic substrates and reaction products.

m/z Retention m/z Retention + + Annotation Substrate [M] or time CID spectrum Product [M] or time CID spectrum + + Criteria [M+H] (min) [M+H] (min) PsREPI 300 (4), 269 (5), 254 (2), 237 (13), 298 (100), 283 (70), 282 (6), 268 225 (4), 209 (13), 197 (4), 181 (4), N-Methyl-1,2 (3), 255.4 (4), 254.9 (3), 254.4 179 (3), 175 (19), 163 (7), 160 (3), 298.2 1.48 (R)-N-Methylcoclaurine 300.2 2.30 Inferred dehydrococlaurine (10), 206.7 (2), 202.2 (2), 198 159.8 (3), 145 (6), 143 (17), 137 (2), 190 (3), 189.8 (2), 107 (2) (12), 131 (6), 127 (2), 121 (8), 107 (100) 300 (4), 269 (5), 254 (2), 237 (13), 225 (4), 209 (13), 197 (4), 181 (4), 300 (4), 269 (5), 254 (2), 237 179 (3), 175 (19), 163 (7), 160 (3), (R)-N-Methylcoclaurine 300.2 2.30 Inferred (13), 225 (4), 209 (13), 197 (4), 159.8 (3), 145 (6), 143 (17), 137 (S)-N- 181 (4), 179 (3), 175 (19), 163 (12), 131 (6), 127 (2), 121 (8), 107 300.2 2.30 Methylcoclaurine (7), 160 (3), 159.8 (3), 145 (6), (100) 143 (17), 137 (12), 131 (6), 127 298 (100), 283 (70), 282 (6), 268 (2), 121 (8), 107 (100) (3), 255.4 (4), 254.9 (3), 254.4 N-Methyl-1,2-dehydrococlaurine 298.2 1.48 Inferred (10), 206.7 (2), 202.2 (2), 198 (2), 190 (3), 189.8 (2), 107 (2) 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 328 (100), 313 (46), 312 (42), 1,2 (100), 179 (2), 178 (1), 177 (8), Authentic 328.2 2.50 296 (3), 284 (19), 267 (4), 266 (R)-Reticuline 330.2 2.82 Dehydroreticuline 175 (22), 163 (1), 160 (2), 151 (5), Standard (2), 249 (3), 190 (4), 162 (2) 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3) 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 330 (1), 299 (2), 267 (3), 239 (3), (100), 179 (2), 178 (1), 177 (8), Authentic 235 (2), 227 (2), 207 (3), 192 (R)-Reticuline 330.2 2.82 175 (22), 163 (1), 160 (2), 151 (5), Standard (100), 179 (2), 178 (1), 177 (8), (S)-Reticuline 330.2 2.82 149 (1), 143 (25), 137 (42), 119 175 (22), 163 (1), 160 (2), 151 (1), 115 (3), 44 (3) (5), 149 (1), 143 (25), 137 (42), 328 (100), 313 (46), 312 (42), 296 119 (1), 115 (3), 44 (3) Authentic 1,2-Dehydroreticuline 328.2 2.50 (3), 284 (19), 267 (4), 266 (2), 249 Standard (3), 190 (4), 162 (2) 328 (100), 313 (44), 312 (60), 298 344 (5), 263 (2), 253 (4), 225 (2), α-Hydroxy-1,2 (6), 296 (5), 284 (23), 267 (6), 249 NMR 192 (100), 177 (95), 162 (4), 151 dehydroreticuline 344.2 2.33 α-Hydroxyreticuline-1 346.2-1 1.16 (3), 237 (3), 204 (3), 192 (18), 191 structural (67), 149 (26), 137 (8), 123 (2), 1 (4), 190 (12), 176 (5), 162 (4), 151 elucidation 119 (8), 95 (2), 90 (5) (6), 137 (9) 328 (100), 313 (45), 312 (57), 298 344 (5), 263 (2), 253 (4), 225 (2), α-Hydroxy-1,2- (7), 296 (5), 284 (20), 267 (6), 249 NMR 192 (100), 177 (95), 162 (4), 151 dehydroreticuline- 344.2 2.60 α-Hydroxyreticuline-2 346.2-2 1.53 (4), 237 (3), 204 (3), 192 (6), 191 structural (67), 149 (26), 137 (8), 123 (2), 2 (4), 190 (11), 176 (5), 162 (2), 151 elucidation 119 (8), 95 (2), 90 (5) (3), 137 (5) PsDRS

179

300 (4), 269 (5), 254 (2), 237 (13), 225 (4), 209 (13), 197 (4), 298 (100), 283 (70), 282 (6), 268 (S)-N- 181 (4), 179 (3), 175 (19), 163 (3), 255.4 (4), 254.9 (3), 254.4 300.2 2.30 Dehydro-N-methylcoclaurine 298.2 1.48 Inferred Methylcoclaurine (7), 160 (3), 159.8 (3), 145 (6), (10), 206.7 (2), 202.2 (2), 198 (2), 143 (17), 137 (12), 131 (6), 127 190 (3), 189.8 (2), 107 (2) (2), 121 (8), 107 (100) 316 (17), 301 (5), 300 (7), 270 (3), 253 (4), 227 (3), 225 (6), 207 (R,S)-6-O (3), 195 (5), 192 (100), 179 (6), 6-O-Methyl-1,2-dehydro- 314 (100), 299 (71), 298 (100), 284 Methyllaudanosoli 316.2 1.50 1.00 Inferred 178 (4), 177 (9), 175 (23), 161 laudanosoline 314.2 (4), 165 (2), 123 (3) ne (9), 152 (4), 143 (34), 137 (16), 123 (65), 115 (8) 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 328 (100), 313 (46), 312 (42), 296 (100), 179 (2), 178 (1), 177 (8), Authentic (S)-Reticuline 330.2 2.82 1,2-Dehydroreticuline 328.2 2.50 (3), 284 (19), 267 (4), 266 (2), 249 175 (22), 163 (1), 160 (2), 151 Standard (3), 190 (4), 162 (2) (5), 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3) 342 (100), 327 (34), 326 (17), 325 192 (100), 177 (19), 151 (15), (13), 314 (15), 310 (14), 299 (19), (S)-Codamine 344.2 3.07 1,2-Dehydrocodamine 342.2 2.50 Inferred 150 (12), 43 (25), 136 (2) 298 (24), 192 (12), 175 (12), 161 (12), 151 (14), 150 (14) PrDRS 300 (4), 269 (5), 254 (2), 237 (13), 225 (4), 209 (13), 197 (4), 298 (100), 283 (70), 282 (6), 268 (S)-N 181 (4), 179 (3), 175 (19), 163 (3), 255.4 (4), 254.9 (3), 254.4 300.2 2.30 N-Methyl-1,2-dehydrococlaurine 298.2 1.48 Inferred Methylcoclaurine (7), 160 (3), 159.8 (3), 145 (6), (10), 206.7 (2), 202.2 (2), 198 (2), 143 (17), 137 (12), 131 (6), 127 190 (3), 189.8 (2), 107 (2) (2), 121 (8), 107 (100) 316 (17), 301 (5), 300 (7), 270 (3), 253 (4), 227 (3), 225 (6), 207 (R,S)-6-O (3), 195 (5), 192 (100), 179 (6), 6-O-Methyl-1,2-dehydro- 314 (100), 299 (71), 298 (100), 284 Methyllaudanosoli 316.2 1.50 314.2 1.00 Inferred 178 (4), 177 (9), 175 (23), 161 laudanosoline (4), 165 (2), 123 (3) ne (9), 152 (4), 143 (34), 137 (16), 123 (65), 115 (8) 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 328 (100), 313 (46), 312 (42), 296 (100), 179 (2), 178 (1), 177 (8), Authentic (S)-Reticuline 330.2 2.82 1,2-Dehydroreticuline 328.2 2.50 (3), 284 (19), 267 (4), 266 (2), 249 175 (22), 163 (1), 160 (2), 151 Standard (3), 190 (4), 162 (2) (5), 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3) 342 (100), 327 (34), 326 (17), 325 192 (100), 177 (19), 151 (15), (13), 314 (15), 310 (14), 299 (19), (S)-Codamine 344.2 3.07 1,2-Dehydrocodamine 342.2 2.50 Inferred 150 (12), 43 (25), 136 (2) 298 (24), 192 (12), 175 (12), 161 (12), 151 (14), 150 (14) PsDRR1 300 (4), 269 (5), 254 (2), 237 (13), 298 (100), 283 (70), 282 (6), 268 225 (4), 209 (13), 197 (4), 181 (4), N-Methyl-1,2 (3), 255.4 (4), 254.9 (3), 254.4 179 (3), 175 (19), 163 (7), 160 (3), 298.2 1.48 (R)-N-Methylcoclaurine 300.2 2.30 Inferred dehydrococlaurine (10), 206.7 (2), 202.2 (2), 198 159.8 (3), 145 (6), 143 (17), 137 (2), 190 (3), 189.8 (2), 107 (2) (12), 131 (6), 127 (2), 121 (8), 107 (100)

180

330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 328 (100), 313 (46), 312 (42), (100), 179 (2), 178 (1), 177 (8), Authentic Dehydroreticuline 328.2 2.50 296 (3), 284 (19), 267 (4), 266 (R)-Reticuline 330.2 2.82 175 (22), 163 (1), 160 (2), 151 (5), Standard (2), 249 (3), 190 (4), 162 (2) 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3) 328 (100), 313 (44), 312 (60), 298 344 (5), 263 (2), 253 (4), 225 (2), α-Hydroxy-1,2 (6), 296 (5), 284 (23), 267 (6), 249 NMR 192 (100), 177 (95), 162 (4), 151 dehydroreticuline 344.2 2.33 α-Hydroxyreticuline-1 346.2-1 1.16 (3), 237 (3), 204 (3), 192 (18), 191 structural (67), 149 (26), 137 (8), 123 (2), 1 (4), 190 (12), 176 (5), 162 (4), 151 elucidation 119 (8), 95 (2), 90 (5) (6), 137 (9) 328 (100), 313 (45), 312 (57), 298 344 (5), 263 (2), 253 (4), 225 (2), α-Hydroxy-1,2- (7), 296 (5), 284 (20), 267 (6), 249 NMR 192 (100), 177 (95), 162 (4), 151 dehydroreticuline- 344.2 2.60 α-Hydroxyreticuline-2 346.2-2 1.53 (4), 237 (3), 204 (3), 192 (6), 191 structural (67), 149 (26), 137 (8), 123 (2), 2 (4), 190 (11), 176 (5), 162 (2), 151 elucidation 119 (8), 95 (2), 90 (5) (3), 137 (5) PrDRR 300 (4), 269 (5), 254 (2), 237 (13), 298 (100), 283 (70), 282 (6), 268 225 (4), 209 (13), 197 (4), 181 (4), N-Methyl-1,2 (3), 255.4 (4), 254.9 (3), 254.4 179 (3), 175 (19), 163 (7), 160 (3), 298.2 1.48 (R)-N-Methylcoclaurine 300.2 2.30 Inferred dehydrococlaurine (10), 206.7 (2), 202.2 (2), 198 159.8 (3), 145 (6), 143 (17), 137 (2), 190 (3), 189.8 (2), 107 (2) (12), 131 (6), 127 (2), 121 (8), 107 (100) 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 328 (100), 313 (46), 312 (42), 1,2 (100), 179 (2), 178 (1), 177 (8), Authentic 328.2 2.50 296 (3), 284 (19), 267 (4), 266 (R)-Reticuline 330.2 2.82 Dehydroreticuline 175 (22), 163 (1), 160 (2), 151 (5), Standard (2), 249 (3), 190 (4), 162 (2) 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3) PsPODA 356 (59), 338 (11), 336 (1), 336 (1), 325 (5), 323 (4), 322 (2), 307 (5), 306 (4), 297 (5), 295 (6), 293 (2), 277 (19), 275 (6), 269 (2), 269 370 (61), 352 (4), 352 (4), 339 (2), 267 (2), 265 (6), 264 (1), 263 (1), 321 (8), 311 (5), 309 (2), 306 (3), 253 (2), 251 (1), 249 (11), 247 (2), 291 (15), 290 (1), 283 (2), (1), 239 (2), 237 (3), 235 (6), 234 281 (5), 277 (2), 267 (4), 263 (6), (1), 223 (2), 217 (1), 208 (17), 207 Cryptopine 370.2 263 (5), 253 (1), 222 (9), 206 O-desmethylcryptopine 356.2 Inferred (2), 206 (3), 195 (4), 194 (1), 192 (11), 205 (61), 204 (100), 194 (6), 191 (97), 190 (100), 189 (2), (7), 193 (5), 190 (20), 188 (1), 188 (2), 180 (9), 179 (8), 178 (1), 178 (1), 175 (15), 165 (93), 163 177 (2), 176 (8), 175 (12), 165 (1), 151 (2), 150 (11), 149 (15) (14), 163 (3), 161 (1), 160 (1), 151 (62), 149 (16), 147 (3), 137 (3), 135 (4), 119 (6), 107 (2), 95 (2), 91 (3), 58 (1), 44 (2) PsP7ODM 340 (86), 325 (9), 324 (72), 310 NMR (2), 308 (1), 296 (13), 203 (5), 326 (50), 310 (75), 188 (100), 157 Papaverine 340.2 Pacodine 326.2 structural 202 (100), 187 (3), 175 (1), 171 (50) Elucidation (16), 123 (2)

181

A.2. Primers used to amplify sequences for (A) heterologous gene expression in E. coli, (B) virus-induced gene silencing, and (C) real-time quantitative PCR (Chapter 3).

A. Primers used to amplify open reading frames for ligation into pQE30.

Gene Forward Primer (5’ – 3’) Reverse Primer (5’ – 3’)

T6ODM GCGCGGATCCATGGAGAAAGCAAAACTT GCGCCTGCAGCACAACGCACTTTCGAGA

PODA GCGCGGATCCATGGAGACAGCAAAACTT GCGCCTGCAGAGAGTCAAAAAGCAATGA

CODM GCGCGGATCCATGGAGACACCAATACTT GCGCCTGCAGGCACCATATGAATTCTTC

DIOX4 GCGGATCCATGGAGGCACCAAAACTA GCCTGCAGCTAGTCAACCTGAACAAT

DIOX5 GCGCATGCATGGAGACATCAAAACTA GCCTGCAGATTGCTTATATGGGTTGA

DIOX6 GCGGATCCATGGAGACACCAACACTTATG GCCTCGAGTCACATCCTCATGCAGTC

B. Primers used to amplify conserved ORF (*) or gene-specific 3’UTR (†) sequences for ligation into pTRV2.

Gene Forward Primer (5’ – 3’) Reverse Primer (5’ – 3’)

All ODDs * GCGCGGATCCCCTTGTCCTCAACCAAAT GCGCCTCGAGTCCACTTTTAAACAAAGC

T6ODM † GCGCGGATCCCGACGTGATTGCATGTCA GCGCCTCGAGCACAACGCACTTTCGAGA

CODM † GCGCGGATCCGTGAGAAAGTGTGAACAT GCGCCTCGAGCAATCCAATACATTATTT

C. Primers used for real-time quantitative PCR.

Gene Forward Primer (5’ – 3’) Reverse Primer (5’ – 3’)

T6ODM TTGAGGCACAAATGAGAAAATTGA CACAACGCACTTTCGAGAAATTAC

CODM TTGTGCTTAAATTTCGTGGATGAC TGATTACATCACTTGACCCAAACAG

182

A.3. Oligonucleotide primers used to the amplify (A) P7ODM open reading frame for ligation into pET47b, and for (B) qRT-PCR analysis (Chapter 4).

A. Primers used to amplify open reading frames for ligation into pET47b.

Gene Forward Primer (5’ – 3’) Reverse Primer (5’ – 3’)

P7ODM ggtaccagATGGAGAAAGCAAAACTTATGAAGC gagctcTCACATCCTCATGCAGTCAAG

B. Primers used for real-time quantitative PCR.

Gene Forward Primer (5’ – 3’) Reverse Primer (5’ – 3’)

P7ODM TTGGTACCCAGCAAATAACAA CAATAGTTTGACTTAATCGCCAA

CODM TTGTGCTTAAATTTCGTGGATGAC TGATTACATCACTTGACCCAAACAG

T6ODM TTGAGGCACAAATGAGAAAATTGA CACAACGCACTTTCGAGAAATTAC

Elongation TGCTCCAGTTCTTGACTGTC GTCTCTACAACCATGGGCTTG factor 1-a

Polyubiquitin GGGAACACAAACGACACCAAA TCGTCTTCGTGGTGGTAACTAGAG 10

183

A.4. Primers used for (A) the construction of expression vectors, (B) the construction of pTRV vectors, and (C) qRT-PCR analysis of opium poppy organs and plants subjected to VIGS (Chapter 5). A. Primers used to assemble expression constructs

Expression Restriction Strand Sequence (5’ – 3’) construct sites

NotI PrDRS Synthetic gene SpeI NotI PsDRS Synthetic gene SpeI NotI PsREPI Synthetic gene SpeI Forward KpnI GGTACCAGATGGAGAGTAGTGGTGTACCAGTAATC PsDRR Reverse SacI GAGCTCTCAAGCTTCATCATCCCACA Forward KpnI GGTACCAGATGGTGAACACTGGTGTACCTGT PsDRR2 Reverse NotI GCGGCCGCTCAAGCAGCTTTTTCATCCC Forward KpnI GGTACCAGATGGATAGTAGTGGTGTACCTGTAATCC PrDRR Reverse BamHI GGATCCTCAAACTTGGTCATCCCATAACTC KpnI CjCNMT Synthetic gene XhoI

B. Primers used to assemble pTRV2 constructs

pTRV2 Restriction Strand Sequence (5’ – 3’) construct sites

Forward BamHI GGATCCCATCAGTTCCATGCTCTGGT REPI-a Reverse XhoI GGTACCGGGCTCATCTCCACTTGATT Forward BamHI GGATCCCATCACTTCCAAGCTCTGGT COR1.1 Reverse KpnI GGTACCGGGCTCATCTCCACTTGAT Forward EcoRI GAATTCCCTACATACTGTATTGGGTTGAATCATG REPI-b Reverse KpnI GGTACCTAACGGGATAGGACGGTTT

184

C. Primers used for qRT-PCR analysis

Gene Strand Primer sequence (5’–3’)

Forward CAGGCAATGGTGGAGTTGGT NCS1 Reverse CCGTGGCACTGCACCTAGA Forward TCCGAATTGGCATCGAAACT 6OMT Reverse CGGAACAGACGGTCTTCGTT Forward GGTTGATACCGGACCAAGAGATT CNMT Reverse CCCCATTGGAGACGCTTTT Forward GGAACGACTTCGAGTTGATACCA NMCH Reverse CAAGGGCACACCAGGACATA Forward TGCAGGCTTTCGGTCTCATA 4'OMT2 Reverse GAAGGCTTCAATGACTGACTGAATAG Forward CCAGGCAATCATCAAAGAATCA REPI Reverse CACAATCTTCGCCGCTCAGT Forward CACAATGGAAAGTTCGTTGATACC SalSyn Reverse TGGCTAATTCTACACCTCCACAAA Forward TGATAAGTGACATTGGAGAGGATTCA SalR Reverse TTGGGCTTCTGGTTTTTCGT Forward TCCGCGAGTGTCCCTAAGG SalAT Reverse TTCCACCACAGTCAAACATGTTC Forward TTGAGGCACAAATGAGAAAATTGA T6ODM Reverse CACAACGCACTTTCGAGAAATTAC Forward ACTCTCAGTTCCGGCATTCG COR1.1 Reverse CCTTTTACCATTGTTTCAGCTGTTC Forward TTGTGCTTAAATTTCGTGGATGAC CODM Reverse TGATTACATCACTTGACCCAAACAG Forward TGCTCCAGTTCTTGACTGTC Elf1a Reverse GTCTCTACAACCATGGGCTTG Forward GGGAACACAAACGACACCAAA Polyubiquitin 10 Reverse TCGTCTTCGTGGTGGTAACTAGAG Forward TCTCAACCCAAAGGCTAATCG Actin Reverse CCCCAGAATCCAAGACAATACC

185

A.5. (A) Mass spectrometry data for substrates and products of enzymatic reactions in ascending order of m/z (Chapter 3).

Substrate Product Product CID Substrate m/z Substrate CID spectrum Product m/z Reaction + + spectrum [M+H] [M+H]

T6ODM Oripavine Morphinone 362 (100), 284 (13), 256 (10), 245 (2), 241 (1), 227 249 (3), 237 (2), 234 (4), (3), 227 (3), 225 (2), 215 223 (1), 221 (4), 218 (8), 362.2 (3), 213 (4), 203 (1), 199 298.2 217 (1), 209 (1), 195 (2), [M+H+BM O-Demethylation (8), 197 (4), 187 (2), 185 189 (1), 58 (100), 56 (1), E]* (27), 181 (9), 173 (6), 171 44 (2) (2), 157 (3), 89 (2), 87 (1), 61 (2), 44 (15)

Thebaine Neopinone 376 (100), 298 (8), 272 (1), 312 (2), 281 (1), 266 (4), 270 (12), 259 (1), 242 (3), 251 (11), 249 (2), 239 241 (11), 229 (1), 227 (3), 376.2 (1), 234 (1), 223 (1), 221 217 (1), 213 (12), 209 (2), 312.2 [M+H+BM O-Demethylation (5), 218 (4), 205 (2), 195 201 (1), 201 (3), 199 (25), E]* (2), 191 (1), 177 (2), 58 187 (3), 185 (3), 185 (5), (100), 56 (1), 44 (1) 181 (5), 171 (3), 58 (4), 44 (9)

Papaverine

340 (86), 325 (9), 324 (72), 310 (2), 308 (1), 326 (100), 310 (99), 188 340.2 296 (13), 203 (5), 202 326.2 O-Demethylation (11), 187.6 (42), 156 (40) (100), 187 (3), 175 (1), 171 (16), 123 (2)

R1=H, R 2=CH 3=Isopacodine R1=CH 3, R2=H=Pacodine ‡

186

Canadine

340 (10), 324 (1), 176 340.2 (100), 174 (3), 165 (3), 326.2 326 (6), 176 (100), 149 (7) O-Demethylation 149 (7), 119 (1)

R1=H, R 2=CH 3=Nandinine R1=CH 3, R2=H=Tetrahydro thalifendine ‡

Tetrahydropalmatine

356 (10), 340 (2), 308 342 (11), 192 (3), 178 (1), 192 (100), 190 (3), 356.2 342.2 (100), 176 (2), 175 (4), 165 O-Demethylation 177 (2), 176 (1), 165 (2), 163 (3), 151 (4) (18), 151 (1), 150 (4)

R1=H, R 2=CH 3=Corypalmine R1=CH 3, R2=H=Tetrahydro columbamine ‡

187

370 (61), 352 (4), 352 Cryptopine (4), 339 (1), 321 (8), 311 (5), 309 (2), 306 (2), 291 (15), 290 (1), 283 (2), 281 (5), 277 (2), 267 (4), 356 (15), 323 (11), 277 263 (6), 263 (5), 253 (1), 370.2 356.2 (13), 191 (100), 190 (66), O-Demethylation 222 (9), 206 (11), 205 151 (23), 149 (11) (61), 204 (100), 194 (7), 193 (5), 190 (20), 188 R1=H, R 2=CH 3=Izmirine (1), 178 (1), 175 (15), R1=CH 3, R2=H=Not reported in 165 (93), 163 (1), 151 literature (2), 150 (11), 149 (15) ‡ 370 (39), 352 (23), 350 356 (54), 338 (12), 326 (3), (1), 339 (1), 337 (6), 336 325 (1), 323 (8), 322 (6), (5), 322 (4), 321 (6), 320 308 (2), 307 (1), 306 (1), (3), 311 (1), 309 (4), 308 Allocryptopine 297 (2), 295 (3), 293 (1), (2), 307 (1), 306 (7), 294 292 (3), 280 (1), 277 (2), (3), 292 (1), 291 (3), 290 275 (22), 267 (2), 264 (1), (30), 283 (2), 281 (2), 263 (5), 263 (2), 247 (9), 281 (1), 278 (2), 276 (2), 237 (2), 237 (1), 235 (2), 370.2 266 (1), 253 (1), 252 (1), 356.2 O-Demethylation 235 (4), 223 (1), 207 (1), 206 (29), 204 (2), 204 206 (38), 195 (5), 190 (15), (1), 193 (1), 192 (3), 191 189 (88), 188 (100), 178 (6), 190 (8), 189 (31), R1=H, R 2=CH 3=Hunnemanine (6), 177 (11), 177 (8), 175 188 (100), 181 (21), 179 R1=CH 3, R2=H=Thalictricine (3), 167 (1), 163 (1), 161 (1), 178 (3), 177 (2), 176 ‡ (1), 159 (2), 151 (9), 149 (2), 166 (6), 165 (11), (39), 135 (3), 121 (1), 119 163 (2), 153 (5), 151 (9), (1) 135 (3), 44 (1) PODA Oripavine Morphinone 362 (100), 284 (13), 256 (10), 245 (2), 241 (1), 227 249 (3), 237 (2), 234 (4), (3), 227 (3), 225 (2), 215 223 (1), 221 (4), 218 (8), 362.2 (3), 213 (4), 203 (1), 199 298.2 217 (1), 209 (1), 195 (2), [M+H+BM O-Demethylation (8), 197 (4), 187 (2), 185 189 (1), 58 (100), 56 (1), E]* (27), 181 (9), 173 (6), 171 44 (2) (2), 157 (3), 89 (2), 87 (1), 61 (2), 44 (15)

188

Stylopine Demethylenestylopine

324 (18), 307 (1), 277 312 14), 176 (3), 164 (100), O,O- (1), 188 (1), 176 (100), 162 (5), 149 (6), 146 (1), 324.2 312.2 Demethyleneatio 174 (4), 149 (29), 135 137 (8), 135 (1), 118 (2), 91 n (1), 119 (5), 91 (4) (2)

Berberine Demethyleneberberine

336 (44), 321 (65), 320 O,O- (100), 318 (2), 306 (24), 324 (65), 309 (30), 308 336.2 [M+] 324.2 [M+] Demethyleneatio 304 (17), 292 (90), 291 (100), 294 (11), 280 (85) n (3), 278 (6), 275 (7)

Papaverine

340 (86), 325 (9), 324 (72), 310 (2), 308 (1), 326 (100), 310 (99), 188 340.2 296 (13), 203 (5), 202 326.2 O-Demethylation (11), 187.6 (42), 156 (40) (100), 187 (3), 175 (1), 171 (16), 123 (2)

R1=H, R 2=CH 3=Isopacodine R1=CH 3, R2=H=Pacodine ‡

189

Canadine Demethylenecanadine

340 (10), 324 (1), 176 328 (5), 192 (1), 177 (1), O,O 340.2 (100), 174 (3), 165 (3), 328.2 165 (2), 164 (100), 150 (2), Demethyleneatio 149 (7), 119 (1) 137 (2) n

Tetrahydropapaverine 327 (2), 312 (2), 297 (2), 282 (2), 281 (2), 281 (1), 296 (6), 281 (3), 192 266 (2), 253 (2), 250 (1), (100), 189 (34), 177 (3), 192 (3), 189 (1), 189 (2), 176 (2), 174 (17), 165 344.2 330.2 178 (100), 175 (13), 174 O-Demethylation (4), 159 (4), 158 (12), (3), 163 (2), 158 (1), 151 151 (51), 150 (1), 148 (35), 143 (34), 137 (5), 115 (1), 144 (1), 136 (2), 129 R1=H, R 2=CH 3=Not reported in (7) (1) literature R1=CH 3, R2=H=Not reported in literature ‡ 354 (82), 336 (7), 323 342 (54), 324 (14), 322 (4), (3), 321 (2), 320 (2), 311 Demethyleneprotopine 311 (2), 309 (2), 308 (1), Protopine (3), 305 (2), 304 (1), 293 306 (1), 293 (4), 283 (3), (3), 275 (15), 267 (2), 281 (2), 263 (24), 255 (2), 265 (3), 263 (2), 262 (1), 253 (2), 235 (13), 223 (1), 253 (2), 247 (10), 237 O,O- 207 (3), 206 (2), 206 (5), 354.2 (2), 235 (1), 217 (2), 206 342.2 Demethyleneatio 195 (1), 194 (13), 188 (3), (21), 204 (2), 195 (4), n 178 (7), 177 (55), 176 189 (100), 188 (91), 178 (100), 175 (5), 166 (2), 165 (3), 177 (9), 175 (2), 175 (29), 164 (2), 163 (1), 149 (9), 165 (12), 164 (10), (9), 147 (3), 137 (8), 135 163 (3), 159 (1), 147 (6), 107 (3) (49), 135 (2), 119 (1)

190

Tetrahydropalmatine

356 (10), 340 (2), 308 (1), 192 (100), 190 (3), 342 (3), 327 (6), 192 (100), 356.2 342.2 O-Demethylation 177 (2), 176 (1), 165 190 (3), 165 (12), 150 (2) (18), 151 (1), 150 (4)

R1=H, R 2=CH 3=Tetrahydro palmatrubine

R1=CH 3, R2=H=Corydalmine ‡ 356 (59), 338 (11), 336 (1), 336 (1), 325 (5), 323 (4), 322 (2), 307 (5), 306 (4), 297 (5), 295 (6), 293 (2), 277 (19), 275 (6), 269 (2), 370 (61), 352 (4), 352 269 (2), 267 (2), 265 (6), Cryptopine (4), 339 (1), 321 (8), 311 264 (1), 263 (3), 253 (2), (5), 309 (2), 306 (2), 291 251 (1), 249 (11), 247 (1), (15), 290 (1), 283 (2), 239 (2), 237 (3), 235 (6), 281 (5), 277 (2), 267 (4), 234 (1), 223 (2), 217 (1), 263 (6), 263 (5), 253 (1), 370.2 356.2 208 (17), 207 (2), 206 (3), O-Demethylation 222 (9), 206 (11), 205 195 (4), 194 (1), 192 (6), (61), 204 (100), 194 (7), 191 (97), 190 (100), 189 193 (5), 190 (20), 188 R1=H, R 2=CH 3=Izmirine (2), 188 (2), 180 (9), 179 (1), 178 (1), 175 (15), R1=CH 3, R2=H=Not reported in (8), 178 (1), 177 (2), 176 165 (93), 163 (1), 151 literature (8), 175 (12), 165 (14), 163 (2), 150 (11), 149 (15) ‡ (3), 161 (1), 160 (1), 151 (62), 149 (16), 147 (3), 137 (3), 135 (4), 119 (6), 107 (2), 95 (2), 91 (3), 58 (1), 44 (2)

191

370 (39), 352 (23), 350 (1), 339 (1), 337 (6), 336 358 (25), 340 (14), 338 (1), (5), 322 (4), 321 (6), 320 327 (2), 325 (2), 324 (4), (3), 311 (1), 309 (4), 308 309 (4), 308 (2), 299 (3), Allocryptopine Vaillantine (2), 307 (1), 306 (7), 294 297 (2), 296 (1), 294 (8),

(3), 292 (1), 291 (3), 290 291 (2), 281 (1), 280 (2), (30), 283 (2), 281 (2), 278 (32), 271 (1), 266 (3), 281 (1), 278 (2), 276 (2), 265 (1), 263 (1), 262 (1), O,O- 370.2 266 (1), 253 (1), 252 (1), 358.2 253 (1), 250 (1), 222 (1), Demethyleneatio 206 (29), 204 (2), 204 194 (24), 191 (3), 188 (0), n (1), 193 (1), 192 (3), 191 181 (12), 178 (10), 177 (6), 190 (8), 189 (31), (11), 176 (100), 175 (1), 188 (100), 181 (21), 179 166 (8), 165 (11), 164 (2), (1), 178 (3), 177 (2), 176 161 (2), 160 (1), 153 (2), (2), 166 (6), 165 (11), 151 (8), 137 (3), 123 (1), 163 (2), 153 (5), 151 (9), 123 (1), 44 (2) 135 (3), 44 (1) CODM 300 (100), 266 (2), 251 (3), 250 (2), 243 (5), 243 286 (100), 268 (4), 229 (3), Codeine (5), 225 (10), 225 (5), Morphine 229 (2), 227 (2), 227 (3), 221 (1), 215 (35), 213 221 (1), 219 (2), 211 (11), (5), 212 (2), 210 (2), 208 209 (8), 201 (20), 199 (5), (3), 201 (1), 200 (1), 199 193 (6), 191 (4), 185 (11), (14), 198 (2), 194 (4), 183 (8), 181 (5), 181 (6), 300.2 193 (3), 187 (15), 185 286.2 178 (2), 173 (11), 168 (1), O-Demethylation (8), 183 (15), 182 (1), 166 (1), 165 (6), 165 (8), 181 (8), 181 (2), 178 (3), 160 (1), 157 (9), 155 (11), 175 (1), 172 (2), 171 (4), 153 (3), 153 (2), 147 (6), 165 (15), 161 (6), 159 145 (6), 127 (1), 123 (2), (2), 155 (4), 155 (6), 153 121 (2), 58 (12), 55 (2), 44 (2), 153 (3), 137 (5), 58 (5) (16), 45 (3), 44 (5) Thebaine Oripavine

312 (2), 281 (1), 266 (4), 252 (1), 249 (2), 249 (2), 251 (11), 249 (2), 239 237 (2), 234 (3), 221 (5), (1), 234 (1), 223 (1), 221 312.2 298.2 218 (9), 217 (2), 209 (1), O-Demethylation (5), 218 (4), 205 (2), 195 206 (1), 195 (2), 189 (3), 58 (2), 191 (1), 177 (2), 58 (100), 56 (1), 44 (1) (100), 56 (1), 44 (1)

192

Scoulerine 3-O-Demethylscoulerine

328 (11), 313 (1), 296 (2), 279 (1), 178 (93), 314 (8), 299 (1), 282 (2), 328.2 176 (3), 163 (4), 151 314.2 178 (3), 164 (80), 151 (2), O-Demethylation (10), 137 (1), 119 (2), 91 137 (4) (1)

Reticuline N-Demethylluxandrine 330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 (100), 179 235 (2), 211 (1), 178 (100), (2), 178 (1), 177 (8), 175 175 (1), 161 (17), 143 (13), 330.2 326.2 O-Demethylation (22), 163 (1), 160 (2), 137 (24), 123 (4), 122 (1), 151 (5), 149 (1), 143 115 (1), 44 (3) (25), 137 (42), 119 (1), 115 (3), 44 (3)

311 (18), 296 (42), 281 (31), 280 (36), 279 (100), Isocorydine 278 (2), 268 (1), 267 (5), 265 (40), 264 (76), 263 (4), 262 (1), 262 (1), 261 282 (1), 279 (71), 267 (1), (2), 253 (2), 252 (2), 251 266 (3), 265 (5), 264 (100), (24), 250 (2), 249 (5), 251 (2), 248 (3), 248 (3), 342.2 328.2 O-Demethylation 248 (67), 247 (19), 237 247 (1), 237 (5), 236 (15),

(3), 236 (45), 235 (5), 235 (4), 221 (1), 219 (4), 233 (1), 233 (2), 231 (2), 191 (3) R1=H, R 2=CH 3= N- 224 (1), 223 (4), 221 (3), Methyllindcarpine 220 (3), 219 (9), 218 (1), R1=CH 3, R2=H=Not reported in 208 (3), 207 (1), 205 (2), literature 193 (1), 191 (9), 44 (3) ‡

193

Demethyleneprotopine

342 (100), 324 (26), 322 (7), 311 (1), 293 (2), 274 (17), 271 (2), 263 (9), 247 (14), 235 (7), 223 (7), 206 O,O- (33), 194 (21), 190 (12), 342.2 Demethyleneatio 354 (82), 336 (7), 323 189 (70), 188 (91), 178 (3), n (3), 321 (2), 320 (2), 311 177 (38), 176 (74), 165 Protopine (3), 305 (2), 304 (1), 293 (32), 163 (7), 149 (18), 137 (3), 275 (15), 267 (2), (5), 135 (31), 130 (2), 107 265 (3), 263 (2), 262 (1), (1) 253 (2), 247 (10), 237 354.2 (2), 235 (1), 217 (2), 206 (21), 204 (2), 195 (4), 189 (100), 188 (91), 178 Demethyleneprotopine (3), 177 (9), 175 (2), 175 (9), 165 (12), 164 (10), 163 (3), 159 (1), 147 342 (41), 324 (12), 322 (3), (49), 135 (2), 119 (1) 283 (4), 281 (9), 263 (25), 255 (3), 235 (6), 223 (3), O,O- 206 (4), 194 (10), 188 (2), 342.2 Demethyleneatio 178 (4), 177 (62), 176 n (100), 175 (5), 165 (35), 164 (3), 149 (6), 137 (10), 135 (9), 107 (4)

Tetrahydropalmatine

356 (10), 340 (2), 308 342 (8), 326 (2), 308 (2), (1), 192 (100), 190 (3), 356.2 342.2 178 (100), 163 (3), 163 (6), O-Demethylation 177 (2), 176 (1), 165 151 (5), 150 (1), 150 (2), (18), 151 (1), 150 (4)

R1=H, R 2=CH 3=Corypalmine R1=CH 3, R2=H=Tetrahydro columbamine ‡

194

356 (59), 338 (11), 336 (1), 336 (1), 325 (5), 323 344 (45), 326 (16), 324 (2), (4), 322 (2), 307 (5), 306 313 (4), 311 (3), 310 (4), (4), 297 (5), 295 (6), 293 301 (1), 295 (8), 294 (3), O-Demethylcryptopine (2), 277 (19), 275 (6), 285 (3), 283 (2), 281 (2), 269 (2), 269 (2), 267 (2), 280 (2), 277 (16), 267 (2), 265 (6), 264 (1), 263 (3), 265 (1), 263 (6), 262 (1), 253 (2), 251 (1), 249 257 (2), 253 (5), 251 (2), (11), 247 (1), 239 (2), 249 (9), 241 (3), 239 (2), 237 (3), 235 (6), 234 (1), 235 (5), 225 (2), 223 (3), O,O- 223 (2), 217 (1), 208 356.2 344.2 213 (1), 208 (24), 207 (4), Demethyleneatio (17), 207 (2), 206 (3), 195 (5), 194 (2), 194 (2), n 195 (4), 194 (1), 192 (6), 192 (15), 191 (80), 190 191 (97), 190 (100), 189 R1=H, R 2=CH 3, (100), 189 (3), 182 (1), 180 (2), 188 (2), 180 (9), 179 R3=R4=OH=Demethylene-izmirine (16), 179 (5), 177 (5), 176 (8), 178 (1), 177 (2), 176 R1=H,R 2=CH 3=Izmirine R1= CH 3, R2=H, R 3=R4=OH=Not (9), 175 (3), 165 (2), 164 (8), 175 (12), 165 (14), R1=CH 3,R2=H=Not found in reported in literature (2), 163 (8), 161 (3), 160 163 (3), 161 (1), 160 (1), literature ‡ ‡ (4), 151 (31), 147 (1), 137 151 (62), 149 (16), 147 (12), 135 (15), 123 (2), 119 (3), 137 (3), 135 (4), 119 (2), 91 (2), 58 (1), 44 (3) (6), 107 (2), 95 (2), 91 (3), 58 (1), 44 (2) 370 (61), 352 (4), 352 358 (38), 340 (2), 340 (6), Cryptopine (4), 339 (1), 321 (8), 311 Demethylenecryptopine 327 (3), 325 (4), 324 (3), (5), 309 (2), 306 (2), 291 309 (4), 308 (1), 298 (5), (15), 290 (1), 283 (2), 297 (3), 291 (8), 291 (8), 281 (5), 277 (2), 267 (4), 281 (1), 277 (5), 271 (2), O,O- 263 (6), 263 (5), 253 (1), 263 (3), 255 (1), 225 (4), 370.2 358.2 Demethyleneatio 222 (9), 206 (11), 205 222 (9), 206 (13), 205 (39), n (61), 204 (100), 194 (7), 204 (100), 194 (7), 193 (7), 193 (5), 190 (20), 188 191 (1), 190 (31), 179 (4), (1), 178 (1), 175 (15), 175 (2), 165 (33), 163 (5), 165 (93), 163 (1), 151 153 (3), 151 (5), 137 (11), (2), 150 (11), 149 (15) 135 (11), 44 (3)

195

356 (54), 338 (12), 326 (3), 325 (1), 323 (8), 322 358 (25), 340 (14), 338 (1), (6), 308 (2), 307 (1), 306 327 (2), 325 (2), 324 (4), Allocryptopine (1), 297 (2), 295 (3), 293 Vaillantine 309 (4), 308 (2), 299 (3), (1), 292 (3), 280 (1), 277 297 (2), 296 (1), 294 (8), (2), 275 (22), 267 (2), 291 (2), 281 (1), 280 (2), 264 (1), 263 (5), 263 (2), 278 (32), 271 (1), 266 (3), 247 (9), 237 (2), 237 (1), 265 (1), 263 (1), 262 (1), O,O- 370.2 235 (2), 235 (4), 223 (1), 358.2 253 (1), 250 (1), 222 (1), Demethyleneatio 207 (1), 206 (38), 195 194 (24), 191 (3), 188 (0), n (5), 190 (15), 189 (88), 181 (12), 178 (10), 177 188 (100), 178 (6), 177 (11), 176 (100), 175 (1), (11), 177 (8), 175 (3), 166 (8), 165 (11), 164 (2), 167 (1), 163 (1), 161 (1), 161 (2), 160 (1), 153 (2), 159 (2), 151 (9), 149 151 (8), 137 (3), 123 (1), (39), 135 (3), 121 (1), 123 (1), 44 (2) 119 (1) DIOX6 Scoulerine 3-O-Demethylscoulerine

328 (11), 313 (1), 296 (2), 279 (1), 178 (93), 314 (8), 299 (1), 282 (2), 328.2 176 (3), 163 (4), 151 314.2 178 (3), 164 (80), 151 (2), O-Demethylation (10), 137 (1), 119 (2), 91 137 (4) (1)

Reticuline N-6-O-Demethylreticuline 330 (2), 299 (3), 267 (3), 255 (1), 241 (1), 239 (2), 235 (2), 227 (2), 195 (1), 235 (2), 211 (1), 178 (100), 192 (100), 179 (2), 178 175 (1), 161 (17), 143 (13), 330.2 (1), 177 (8), 175 (20), 316.2 O-Demethylation 137 (24), 123 (4), 122 (1), 163 (1), 160 (3), 151 (5), 115 (1), 44 (3) 149 (1), 143 (22), 137 (40), 119 (1), 115 (3), 91 (1), 44 (3)

* BME, β-mercaptoethanol adduct. ‡ The regio-specificity of this reaction could not be determined from the CID spectrum. The two possibilities are shown.

196

A.5. (B) Mass spectrometry data for selected compounds detected in plants subjected to VIGS and listed in ascending order of m/z (Chapter 3). m/z Compound + CID spectrum Annotation Criteria [M+H] Cheilanthifoline

326 (24), 277 (2), 178 Liscombe et al., 2009; 326.2 (100), 176 (6), 151 (19), 148 Farrow et al., 2012 (5), 119 (4), 91 (2)

Sanguinarine

332 (100), 330 (5), 317 332.2 (12), 304 (18), 302 (6), Authentic Standard 274 (10), 246 (3)

Dihydrosanguinarine

334 (34), 332 (2), 319 334.2 (100), 318 (31), 304 (21), Authentic Standard (8)

N-Methylstylopine

338 (25.32), 190 (100), 188 338.2 Authentic Standard (5), 176 (1), 163 (1)

Sinactine

340 (17.98), 324 (78), 310 (4), 202 (100), 192 Liscombe et al., 2009; 340.2 (56), 165 (16) Farrow et al., 2012

Tetrahydrocolumbamine

342 (9), 326 (2), 294 (4), 342.2 178 (100), 176 (2), 163 (2), Authentic Standard 151 (8)

N-Methylcanadine

354 (100), 338 (6), 324 (2), 306 (2), 292 (2), 190 Liscombe et al., 2009; 354.2 (70), 188 (2), 176 (2), Farrow et al., 2012 165 (2), 149 (2)

354 (100), 339 (4), 336 (7), Papaverubine B 386.2 326 (6), 322 (41), 310 (9), 308 Dolejš and Hanuš, 1967 (4), 305 (14), 295 (14), 294

197

(4), 293 (14), 292 (5), 290 (6), 269 (4), 265 (12), 263 (6), 249 (6), 234 (4), 204 (4), 192 (87), 190 (5), 177 (5), 176 (10), 165 (6), 151 (4), 137 (4)

386 (14), 368 (100), 340 (7), N-Methylporphyroxine 337 (3), 325 (2), 321 (2), 309 (3), 309 (2), 307 (4), 306 (2), 297 (40), 283 (2), 282 (5), 279 (4), 278 (3), 267 (4), 266 (8), 386.2 Dolejš and Hanuš, 1967 222 (4), 220 (5), 206 (58), 204 (4), 191 (2), 177 (3), 176 (2), 165 (7), 163 (3), 135 (2), 59 (2), 44 (2)

Glaudine

400 (4), 351 (5), 339 (19), 309 400.2 (7), 294 (4), 281 (5), 248 (5), Dolejš and Hanuš, 1967 206 (100), 192 (6), 165 (9)

Noscapine

414 (3), 365 (4), 353 (20), 350 414.2 (4), 323 (4), 220 (100), 206 Authentic Standard (4), 179 (8)

198

A.6. 13C, 1H and NMR spectral data for pacodine (Chapter 4). Values are with reference to atom numbers in Figure 4. Carbon (#), Nitrogen (#N) Chemical Shift J (Hz) COSY HSQC HMBC NOESY or Hydrogen Atom (#H) 1 157.36 α, 8, 3 2N ND 3 135.68 3 4 3H 8.16 6.0 4 3 1, 4α, 8α, 4 4 4 121.67 4 5, 3 4H 7.74 5.9 3 4 8α, 5, 3 3, 5 4α 136.04 3, 8 5 106.82 5 4

5H 7.38 R1 5 8α, 7, 4, 6 4, R 1

6 155.76 R1, 8, 5 7 150.74 5, 8 8 109.27 8 8H 7.56 8 1, 4α, 6, 7 2', 6', α 8α 124.73 4, 5, 3, α α 40.07 α 2',6'

αH2 4.54 2', 6' α 1', 1, 8α, 6', 2' 8, 2', 6' 1' 131.81 5', α 2' 113.55 2' 6', 2', α

2' 6.89 1.9 6', α 2' α, 6', 2', 4', 3' 8, α, R2, R4

3' 150.76 R4, 2', 5'

4' 149.54 R2, 2', 5', 6' 5' 113.3 5'

5'H 6.83 8.3 6' 5' 1', 6', 4', 3' R2 6' 121.94 6' α, 2', 5' 6'H 6.71 8.3, 2.0 2', 5', α 6' α, 2', 4' 8, α

R1 56.78 R1

R1H3 4.04 5 R1 6 5

R2 ND

R2H3 ND

R3 56.51 R3

R3H3 3.75 R3 4' 5', 2'

R4 56.38 R4

R4H3 3.75 R4 3' 2'

199

A.7. NMR data used to identify the contaminant reaction product as α-hydroxyreticuline. Data for major and minor peaks corresponding to α-hydroxyreticuline (m/z 346) are provided (Chapter 5).

m/z 346 (α-Hydroxyreticuline-1)

13 1 HSQC HSQC C δ ( C) δ ( H) J HMBC (HC) INT m COSY ROE (HC) (m) / number (ppm) ppm (Hz) (ppm) (ppm) RING ID

1 70.16 4.12 1.00 d 9.19 α, 8 (vw) 8 (vs), 2' (s), 6' (m), NMe (s) 70.16 CH/CH 3 a (w), 3 (vw), 8 (vw), 9 (vw), 10 (vw)

3α 45.52 3.78 1.00 m - 3β, 4α, 4β α (s), 3β (vs), 4α (m), 4β (w) 45.52 CH2 NMe (m)

3β 45.52 3.26 1.00 m - 3α, 4α, 4β (w) 3α (vs), 4β (m), NMe (m) 45.52 CH2 4 (vw), 10 (w)

4 22.28 3.11 1.00 m - 3α, 3β, 4β 3α (m), NMe (m), 5 (s) 22.28 CH2 3 (m), 5 (vw), 9 (w), 10 (m)

4β 22.28 2.99 1.00 m - 3α, 3β (w), 4α, 5 (vw) 3α (w), 3β (m), 5 (s) 22.28 CH2 9 (w) 10 (w)

5 112.07 6.75 1.00 s - 4β (vw), 6-OMe (w) 4α (s), 4β (s), 6-OMe (vs) 112.07 CH/CH 3 4 (s), 6 (s), 7 (vs), 9 (s), 10 (vw) 6 149.25 ------

6-OMe 56.00 3.82 3.00 s - 5 (w) 5 (vs) 56.00 CH/CH 3 6 (vs) 7-OH 145.55 ------

8 116.89 5.62 1.00 s - 1 (vw) 1 (vs), α (vw), 2' (w), 6' (w) 116.89 CH/CH 3 1 (s), 4 (vw), 6 (s), 7 (s), 10 (s) 9 120.53 ------10 122.79 ------1' 133.61 ------

2' 115.34 6.83 1.00 d 1.65 α, 6' α (s), 1 (s), 8 (w) 115.34 CH/CH 3 a (m), 3' (m), 4' (vs), 6' (s) 3'-OH 147.52 ------4' 149.23 ------

4'-OMe 56.07 3.87 3.00 s - 5' (vw) 5' (vs) 56.07 CH/CH 3 4' (vs)

5' 112.17 6.89 1.00 d 8.30 4'-OMe (vw), 6' 4'-OMe (vs) 112.17 CH/CH 3 1' (s), 3' (s), 4' (m), 8.30, 6' 120.56 6.64 1.00 dd 2', 5' α (s), 8 (w), 1 (m) 120.56 CH/CH 3 a (m), 2' (s), 4' (vs), 5' (vw) 1.65

NMe 40.49 2.81 3.00 s - - 1 (s),3β (m), 4a (m) 40.49 CH/CH 3 1 (vs), 3 (vs)

α 75.44 4.56 - d 9.19 1, 2' (vw) 3α (s), 8 (vw), 2' (s), 6' (s) 75.44 CH/CH 3 1 (s), 2' (w) α-OH 75.44 ------

200

m/z 346 (α-Hydroxyreticuline-2)

13 1 HSQC HSQC C δ ( C) δ ( H) J HMBC (HC) INT m COSY ROE (HC) (m) / number ppm ppm (Hz) (ppm) (ppm) RING ID

1 70.34 4.26 1H s (br) - α, 8 (vw) α (m), 8 (s), 2' (vw), NMe (m) 70.34 CH/CH 3 -

3α 49.29 3.30 obsc amb - 3β, 4α, 4β 3β (s), 4α (w) 49.29 CH2 - 12.1, 3β 49.29 2.92 1H ddd 4.64, 3α, 4α, 4β 3α (s), 4β (s) 49.29 CH2 1 (w), 10 (w) 4.64

4α 24.08 2.84 1H m - 3α, 3β, 4β, 5 (vw) 3α (w), 5 (s) 24.08 CH2 5 (vw), 9 (vw), 10 (w) 16.59, 4β 24.08 2.62 1H ddd 4.64, 3α, 3β, 4α, 5 (w) 3β (s), 5(s) 24.08 CH2 3 (w), 5 (w), 9 (vw), 10 (m) 4.64

5 112.01 6.70 1H s - 4α (vw), 4β (w), 6-OMe 4α (s), 4β (s), 6-OMe (vs) 112.01 CH/CH 3 4 (m), 6 (m), 7 (vs), 9 (s) 6 148.80 ------

6-OMe 56.12 3.84 3H s - 5 5 (vs) 56.12 CH/CH 3 6 (vs) 7-OH 145.78 ------

8 116.08 6.32 1H s - 1 (vw) α (s), 1 (m), 6' (vw), 2' (vw) 116.08 CH/CH 3 1 (m), 6 (vs), 7 (m), 10 (vs) 9 121.75 ------10 125.41 ------1' 133.90 ------

2' 114.45 6.64 1H d 1.58 α (w) α (m), 1 (vw), 8 (vw) 114.45 CH/CH 3 α (w), 3' (m), 4' (s), 6' (s) 3'-OH 147.33 ------4' 148.50 ------

4'-OMe 56.06 3.83 3H s - 5' (vw) 5' (vs) 56.06 CH/CH 3 4' (vs)

5' 112.07 6.84 1H d 8.30 4'-OMe (vw), 6' 4'-OMe (vs) 112.07 CH/CH 3 1' (s), 3' (s), 4' (w) 8.30, 6' 118.62 6.61 1H dd α (w), 5' α (m), 8 (vw) 118.62 CH/CH 3 2' (s), 4' (s) 1.58

NMe 43.21 2.83 3H s - - α (w), 1 (m) 43.21 CH/CH 3 1 (vs), 3 (vs)

α 74.92 5.14 1H d 2.54 1, 2' (w), 6' (w) 1 (m), 3α (w), 8 (s), 2' (m), 6' (m), NMe (w) 74.92 CH/CH 3 6' (vw) α-OH 74.92 ------

201

A.8. Primers used to detect pTRV2 in infiltrated plants (Chapter 3 and Chapter 5)

Restriction Region Strand Sequence (5’ – 3’) sites

Forward BamHI GGATCCCATCAGTTCCATGCTCTGGT MCS Reverse XhoI GGTACCGGGCTCATCTCCACTTGATT A.9. Parameters used for multiple-reaction monitoring of benzylisoquinoline alkaloids in opium poppy plants subjected to virus-induced gene silencing (Chapter 3).

Fragmentor Collision Energy Compound Precursor Ion (m/z) Product Ion (m/z) Voltage (V) (eV)

Allocryptopine 370.2 188.2 110 25

Cryptopine 370.2 205.2 110 35

Protopine 354.2 189.2 113 30

Cheilanthifoline 326.2 178.2 113 25

Canadine 340.2 176.2 113 25

O-Demethylcryptopine 356.2 190.2 110 35

Tetrahydrocolumbamine 342.2 178.2 128 25

Stylopine 324.2 176.2 113 32

Laudanosine 358.2 206.1 110 25

N-Methylporphyroxine 386.2 206.2 110 25

Papaverubine B 386.2 354.2 110 25

Glaudine 400.2 206.2 110 25

Sinactine 340.2 192.2 113 25

N-Methylstylopine 338.2 190.2 110 35

Tetrahydropalmatine 356.2 192.2 134 28

202

A.10. Chromatographic and mass spectral data used for the identification and relative quantification of benzylisoquinoline alkaloids in opium poppy plants subjected to virus-induced gene silencing (Chapter 5). Multiple reaction monitoring was used to detect and quantify codamine and laudanine owing to their co-elution.

MRM Retention m/z transitions Annotation Compound + time CID spectrum [M+H] (when Criteria (min) required) 286 (100), 268 (4), 229 (3), 227 (3), 211 (11), 209 (8), 201 (20), 199 (5), 193 (6), 191 (4), 185 (11), 183 (8), 181 Authentic Morphine 286.2 - 1.00 (5), 181 (6), 173 (11), 165 (6), 165 (8), Standard 157 (9), 155 (11), 153 (3), 153 (2), 147 (6), 145 (6), 58 (12), 55 (2), 44 (5) 300 (100), 251 (3), 243 (5), 243 (5), 225 (10), 225 (5), 215 (35), 213 (5), 208 (3), 199 (14), 194 (4), Authentic Codeine 300.2 - 3.34 193 (3), 187 (15), 185 (8), 183 (15), Standard 181 (8), 178 (3), 171 (4), 165 (15), 161 (6), 155 (4), 155 (6), 153 (3), 137 (5), 58 (16), 45 (3), 44 (5)

312 (2), 281 (1), 266 (4), 251 (11), 249 (2), 239 (1), 234 (1), 223 (1), 221 (5), Authentic Thebaine 312.2 - 4.51 218 (4), 205 (2), 195 (2), 191 (1), 177 Standard (2), 58 (100), 56 (1), 44 (1)

330 (1), 299 (2), 267 (3), 239 (3), 235 (2), 227 (2), 207 (3), 192 (100), 179 Authentic Reticuline 330.2 - 3.93 (2), 178 (1), 177 (8), 175 (22), 163 (1), Standard 160 (2), 151 (5), 149 (1), 143 (25), 137 (42), 119 (1), 115 (3), 44 (3)

340 (86), 325 (9), 324 (72), 310 (2), Authentic Papaverine 340.2 - 6.06 308 (1), 296 (13), 203 (5), 202 (100), Standard 187 (3), 175 (1), 171 (16), 123 (2)

344 (2), 313 (2), 298 (11), 282 (22), 192.2 206 (100), 191 (7), 189 (51), 175 (16), Schmidt et Laudanine 344.2 (quantifier) 4.38 163 (5), 158 (10), 151 (18), 150 (16), al., 2007 151 (qualifier) 137 (75), 137 (64)

206.2 192 (100), 177 (19), 151 (15), 150 (12), Codamine 344.2 (quantifier) 4.29 inferred 43 (25), 136 (2) 137.2 (qualifier)

312 (3), 296 (5), 268 (2), 255 (2), 206 (100), 191 (2), 189 (35), 174 (11), 165 Schmidt et Laudanosine 358.2 - 4.75 (6), 164 (7), 161 (2), 159 (4), 158 (6), al., 2007 151 (66), 150 (3), 145 (3)

414 (4), 365 (5), 353 (21), 323 (5), 220 Authentic Noscapine 414.2 - 7.62 (100), 206 (4), 179 (8) Standard

203

A.11. Virus-induced gene silencing of PsREPI in opium poppy using a unique region of the gene. (A) Fragment (grey box) of the PsREPI cDNA used to assemble the pTRV2 construct. The black box represents the coding region, whereas the black lines are the flanking untranslated regions. Arrows show the annealing sites of primers used for qRT- PCR analysis (Appendix 4). (B) Ethidium bromide-stained agarose gels showing the

204

(Continued from previous page) detection of the pTRV2 vector by RT-PCR using total RNA extracted from individual plants infiltrated with Agrobacterium tumefaciens harbouring the pTRV2-REPI-5’ construct or the pTRV2 empty vector control. PCR primers (TRV2-mcs) were designed to anneal to regions flanking the multiple cloning site (mcs) of pTRV2. (C) Relative PsREPI transcript abundance in control (pTRV2) and PsREPI-silenced (pTRV2-REPI-5’) plants. (D) Total ion chromatograms showing the major alkaloid profiles of control (pTRV2) and PsREPI-silenced (pTRV2-REPI-5’) plants. Peak numbers correspond to compounds shown in panel E. (E) Relative abundance of major alkaloids and O-methylated reticuline derivatives in the latex and roots of PsREPI-silenced (pTRV2-REPI-5’) plants compared with controls (pTRV2). (F) Relative abundance of S and R enantiomers of reticuline in PsREPI-silenced (pTRV2-REPI-5’) plants compared with controls (pTRV2). The reference gene for results shown was Elf-1a. Values represent the mean ± standard deviation of 16 biological replicates. Asterisks represent significant differences determined using an unpaired, two-tailed Student t test (p <0.05)

205

A.12. Virus-induced gene silencing of PsCOR1.1 in opium poppy. (A) Fragment (grey box) of the PsCOR1.1 cDNA used to assemble the pTRV2 construct. The black box represents the coding region, whereas the black lines are the flanking untranslated regions. Arrows show the annealing sites of primers used for qRT-PCR analysis (Appendix 4). (B) Ethidium bromide-stained agarose gels showing the detection of the pTRV2 vector by

206

(Continued from previous page) RT-PCR using total RNA extracted from individual plants infiltrated with Agrobacterium tumefaciens harbouring the pTRV2-COR1.1 construct or the pTRV2 empty vector control. PCR primers (TRV2-mcs) were designed to anneal to regions flanking the multiple cloning site (mcs) of pTRV2. (C) Relative PsCOR1.1 transcript abundance in control (pTRV2) and PsCOR1.1-silenced (pTRV2- COR1.1) plants. (D) Total ion chromatograms showing the major alkaloid profiles of control (pTRV2) and PsCOR1.1-silenced (pTRV2-COR1.1) plants. Peak numbers correspond to compounds shown in panel E. (E) Relative abundance of major alkaloids and O-methylated reticuline derivatives in the latex and roots of PsCOR1.1-silenced (pTRV2-COR1.1) plants compared with controls (pTRV2). (F) Relative abundance of S and R enantiomers of reticuline in PsCOR1.1-silenced (pTRV2-COR1.1) plants compared with controls (pTRV2). The reference gene for results shown was Elf-1a. Values represent the mean ± standard deviation of 16 biological replicates. Asterisks represent significant differences determined using an unpaired, two-tailed Student t test (p <0.05).

207

A.13. Virus-induced gene silencing of PsREPI in opium poppy. (A) Fragment (grey box) of the PsREPI cDNA used to assemble the pTRV2 construct. The black box represents the coding region, whereas the black lines are the flanking untranslated regions. Arrows show the annealing sites of primers used for qRT-PCR analysis (Appendix 4). (B) Ethidium bromide-stained agarose gels showing the detection of the pTRV2 vector by

208

(Continued from previous page) RT-PCR using total RNA extracted from individual plants infiltrated with Agrobacterium tumefaciens harbouring the pTRV2-REPI-a construct or the pTRV2 empty vector control. PCR primers (TRV2-mcs) were designed to anneal to regions flanking the multiple cloning site (mcs) of pTRV2. (C) Relative PsREPI transcript abundance in control (pTRV2) and PsREPI-silenced (pTRV2-REPI-a) plants. (D) Total ion chromatograms showing the major alkaloid profiles of control (pTRV2) and PsREPI-silenced (pTRV2-REPI-a) plants. Peak numbers correspond to compounds shown in panel E. (E) Relative abundance of major alkaloids and O- methylated reticuline derivatives in the latex and roots of PsREPI-silenced (pTRV2- REPI-a) plants compared with controls (pTRV2). (F) Relative abundance of S and R enantiomers of reticuline in PsREPI-silenced (pTRV2-REPI-a) plants compared with controls (pTRV2). The reference gene for results shown was Elf-1a. Values represent the mean ± standard deviation of 16 biological replicates. Asterisks represent significant differences determined using an unpaired, two-tailed Student t test (p <0.05).

209

A.14. Nucleotide sequence similarity matrix comparing PsREPI to other benzylisoquinoline alkaloid biosynthetic genes. The DRS and DRR1 components of PsREPI were compared separately to assess the potential for off-target silencing in virus-induced gene-silencing experiments. The matrix was generated using the EMBOSS Needle pair- wise alignment tool (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Values below 45% should be considered to reflect a lack of nucleotide sequence identity. Accession numbers: NCS1, AY860500; 6OMT, KJ526085; CNMT, AB061863; CYP80B3, AF191772; 4’OMT2, AY217334; CYP719B1, EF451150; SalAT, AF339913; SalR, DQ316261; T6ODM, GQ500139; COR1.1, AF108432; CODM, GQ500141.

REPI CYP (PsDRS) AKR (PsDRR1)

domain domain NCS1 33 45 6OMT 40 38 CNMT 38 39 CYP80B3 45 33 4'OMT2 40 37 SalSyn 40 31 SalAT 38 41 SalR 43 34 T6ODM 42 38 COR1.1 31 77 CODM 37 40

210 10/10/2015 RightsLink Printable License

ELSEVIER LICENSE TERMS AND CONDITIONS Oct 11, 2015

This is a License Agreement between Scott Farrow ("You") and Elsevier ("Elsevier") provided by Copyright Clearance Center ("CCC"). The license consists of your order details, the terms and conditions provided by Elsevier, and the payment terms and conditions.

All payments must be made in full to CCC. For payment instructions, please see information listed at the bottom of this form.

Supplier Elsevier Limited The Boulevard,Langford Lane Kidlington,Oxford,OX5 1GB,UK Registered Company Number 1982084 Customer name Scott Farrow

Customer address 2500 University Drive NW

Calgary, AB T2N1N4 License number 3725721275173

License date Oct 11, 2015

Licensed content publisher Elsevier Licensed content publication FEBS Letters

Licensed content title Papaverine 7Odemethylase, a novel 2oxoglutarate/Fe2+ dependent dioxygenase from opium poppy

Licensed content author Scott C. Farrow,Peter J. Facchini Licensed content date 14 September 2015

Licensed content volume 589 number Licensed content issue 19 number Number of pages 6 Start Page 2701

End Page 2706 Type of Use reuse in a thesis/dissertation Portion full article

Format both print and electronic Are you the author of this Yes Elsevier article? Will you be translating? No Title of your Expanding the roles of oxidoreductases in benzylisoquinoline alkaloid thesis/dissertation metabolism Expected completion date Dec 2015 https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 1/6 10/10/2015 RightsLink Printable License Estimated size (number of 200 pages) Elsevier VAT number GB 494 6272 12 Permissions price 0.00 CAD VAT/Local Sales Tax 0.00 CAD / 0.00 GBP Total 0.00 CAD Terms and Conditions INTRODUCTION 1. The publisher for this copyrighted material is Elsevier. By clicking "accept" in connection with completing this licensing transaction, you agree that the following terms and conditions apply to this transaction (along with the Billing and Payment terms and conditions established by Copyright Clearance Center, Inc. ("CCC"), at the time that you opened your Rightslink account and that are available at any time at http://myaccount.copyright.com). GENERAL TERMS 2. Elsevier hereby grants you permission to reproduce the aforementioned material subject to the terms and conditions indicated. 3. Acknowledgement: If any part of the material to be used (for example, figures) has appeared in our publication with credit or acknowledgement to another source, permission must also be sought from that source. If such permission is not obtained then that material may not be included in your publication/copies. Suitable acknowledgement to the source must be made, either as a footnote or in a reference list at the end of your publication, as follows: "Reprinted from Publication title, Vol /edition number, Author(s), Title of article / title of chapter, Pages No., Copyright (Year), with permission from Elsevier [OR APPLICABLE SOCIETY COPYRIGHT OWNER]." Also Lancet special credit "Reprinted from The Lancet, Vol. number, Author(s), Title of article, Pages No., Copyright (Year), with permission from Elsevier." 4. Reproduction of this material is confined to the purpose and/or media for which permission is hereby given. 5. Altering/Modifying Material: Not Permitted. However figures and illustrations may be altered/adapted minimally to serve your work. Any other abbreviations, additions, deletions and/or any other alterations shall be made only with prior written authorization of Elsevier Ltd. (Please contact Elsevier at [email protected]) 6. If the permission fee for the requested use of our material is waived in this instance, please be advised that your future requests for Elsevier materials may attract a fee. 7. Reservation of Rights: Publisher reserves all rights not specifically granted in the combination of (i) the license details provided by you and accepted in the course of this licensing transaction, (ii) these terms and conditions and (iii) CCC's Billing and Payment terms and conditions. 8. License Contingent Upon Payment: While you may exercise the rights licensed immediately upon issuance of the license at the end of the licensing process for the transaction, provided that you have disclosed complete and accurate details of your proposed use, no license is finally effective unless and until full payment is received from you (either by publisher or by CCC) as provided in CCC's Billing and Payment terms and conditions. If full payment is not received on a timely basis, then any license preliminarily granted shall be deemed automatically revoked and shall be void as if never granted. Further, in the event that you breach any of these terms and conditions or any of CCC's Billing and Payment terms and conditions, the license is automatically revoked and shall be void as if never https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 2/6 10/10/2015 RightsLink Printable License granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an unrevoked license, may constitute copyright infringement and publisher reserves the right to take any and all action to protect its copyright in the materials. 9. Warranties: Publisher makes no representations or warranties with respect to the licensed material. 10. Indemnity: You hereby indemnify and agree to hold harmless publisher and CCC, and their respective officers, directors, employees and agents, from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this license. 11. No Transfer of License: This license is personal to you and may not be sublicensed, assigned, or transferred by you to any other person without publisher's written permission. 12. No Amendment Except in Writing: This license may not be amended except in a writing signed by both parties (or, in the case of publisher, by CCC on publisher's behalf). 13. Objection to Contrary Terms: Publisher hereby objects to any terms contained in any purchase order, acknowledgment, check endorsement or other writing prepared by you, which terms are inconsistent with these terms and conditions or CCC's Billing and Payment terms and conditions. These terms and conditions, together with CCC's Billing and Payment terms and conditions (which are incorporated herein), comprise the entire agreement between you and publisher (and CCC) concerning this licensing transaction. In the event of any conflict between your obligations established by these terms and conditions and those established by CCC's Billing and Payment terms and conditions, these terms and conditions shall control. 14. Revocation: Elsevier or Copyright Clearance Center may deny the permissions described in this License at their sole discretion, for any reason or no reason, with a full refund payable to you. Notice of such denial will be made using the contact information provided by you. Failure to receive such notice will not alter or invalidate the denial. In no event will Elsevier or Copyright Clearance Center be responsible or liable for any costs, expenses or damage incurred by you as a result of a denial of your permission request, other than a refund of the amount(s) paid by you to Elsevier and/or Copyright Clearance Center for denied permissions. LIMITED LICENSE The following terms and conditions apply only to specific license types: 15. Translation: This permission is granted for nonexclusive world English rights only unless your license was granted for translation rights. If you licensed translation rights you may only translate this content into the languages you requested. A professional translator must perform all translations and reproduce the content word for word preserving the integrity of the article. 16. Posting licensed content on any Website: The following terms and conditions apply as follows: Licensing material from an Elsevier journal: All content posted to the web site must maintain the copyright information line on the bottom of each image; A hypertext must be included to the Homepage of the journal from which you are licensing at http://www.sciencedirect.com/science/journal/xxxxx or the Elsevier homepage for books at http://www.elsevier.com; Central Storage: This license does not include permission for a scanned version of the material to be stored in a central repository such as that provided by Heron/XanEdu. Licensing material from an Elsevier book: A hypertext link must be included to the Elsevier homepage at http://www.elsevier.com . All content posted to the web site must maintain the copyright information line on the bottom of each image.

https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 3/6 10/10/2015 RightsLink Printable License Posting licensed content on Electronic reserve: In addition to the above the following clauses are applicable: The web site must be passwordprotected and made available only to bona fide students registered on a relevant course. This permission is granted for 1 year only. You may obtain a new license for future website posting. 17. For journal authors: the following clauses are applicable in addition to the above: Preprints: A preprint is an author's own writeup of research results and analysis, it has not been peer reviewed, nor has it had any other value added to it by a publisher (such as formatting, copyright, technical enhancement etc.). Authors can share their preprints anywhere at any time. Preprints should not be added to or enhanced in any way in order to appear more like, or to substitute for, the final versions of articles however authors can update their preprints on arXiv or RePEc with their Accepted Author Manuscript (see below). If accepted for publication, we encourage authors to link from the preprint to their formal publication via its DOI. Millions of researchers have access to the formal publications on ScienceDirect, and so links will help users to find, access, cite and use the best available version. Please note that Cell Press, The Lancet and some societyowned have different preprint policies. Information on these policies is available on the journal homepage. Accepted Author Manuscripts: An accepted author manuscript is the manuscript of an article that has been accepted for publication and which typically includes author incorporated changes suggested during submission, peer review and editorauthor communications. Authors can share their accepted author manuscript:

 immediately via their noncommercial person homepage or blog by updating a preprint in arXiv or RePEc with the accepted manuscript via their research institute or institutional repository for internal institutional uses or as part of an invitationonly research collaboration workgroup directly by providing copies to their students or to research collaborators for their personal use for private scholarly sharing as part of an invitationonly work group on commercial sites with which Elsevier has an agreement  after the embargo period via noncommercial hosting platforms such as their institutional repository via commercial sites with which Elsevier has an agreement

In all cases accepted manuscripts should:

 link to the formal publication via its DOI  bear a CCBYNCND license this is easy to do  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article.

Published journal article (JPA): A published journal article (PJA) is the definitive final record of published research that appears or will appear in the journal and embodies all valueadding publishing activities including peer review coordination, copyediting, formatting, (if relevant) pagination and online enrichment. Policies for sharing publishing journal articles differ for subscription and gold open access https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 4/6 10/10/2015 RightsLink Printable License articles: Subscription Articles: If you are an author, please share a link to your article rather than the fulltext. Millions of researchers have access to the formal publications on ScienceDirect, and so links will help your users to find, access, cite, and use the best available version. Theses and dissertations which contain embedded PJAs as part of the formal submission can be posted publicly by the awarding institution with DOI links back to the formal publications on ScienceDirect. If you are affiliated with a library that subscribes to ScienceDirect you have additional private sharing rights for others' research accessed under that agreement. This includes use for classroom teaching and internal training at the institution (including use in course packs and courseware programs), and inclusion of the article for grant funding purposes. Gold Open Access Articles: May be shared according to the authorselected enduser license and should contain a CrossMark logo, the end user license, and a DOI link to the formal publication on ScienceDirect. Please refer to Elsevier's posting policy for further information. 18. For book authors the following clauses are applicable in addition to the above: Authors are permitted to place a brief summary of their work online only. You are not allowed to download and post the published electronic version of your chapter, nor may you scan the printed edition to create an electronic version. Posting to a repository: Authors are permitted to post a summary of their chapter only in their institution's repository. 19. Thesis/Dissertation: If your license is for use in a thesis/dissertation your thesis may be submitted to your institution in either print or electronic form. Should your thesis be published commercially, please reapply for permission. These requirements include permission for the Library and Archives of Canada to supply single copies, on demand, of the complete thesis and include permission for Proquest/UMI to supply single copies, on demand, of the complete thesis. Should your thesis be published commercially, please reapply for permission. Theses and dissertations which contain embedded PJAs as part of the formal submission can be posted publicly by the awarding institution with DOI links back to the formal publications on ScienceDirect.

Elsevier Open Access Terms and Conditions You can publish open access with Elsevier in hundreds of open access journals or in nearly 2000 established subscription journals that support open access publishing. Permitted third party reuse of these open access articles is defined by the author's choice of Creative Commons user license. See our open access license policy for more information. Terms & Conditions applicable to all Open Access articles published with Elsevier: Any reuse of the article must not represent the author as endorsing the adaptation of the article nor should the article be modified in such a way as to damage the author's honour or reputation. If any changes have been made, such changes must be clearly indicated. The author(s) must be appropriately credited and we ask that you include the end user license and a DOI link to the formal publication on ScienceDirect. If any part of the material to be used (for example, figures) has appeared in our publication with credit or acknowledgement to another source it is the responsibility of the user to ensure their reuse complies with the terms and conditions determined by the rights holder. Additional Terms & Conditions applicable to each Creative Commons user license: CC BY: The CCBY license allows users to copy, to create extracts, abstracts and new works from the Article, to alter and revise the Article and to make commercial use of the Article (including reuse and/or resale of the Article by commercial entities), provided the user gives appropriate credit (with a link to the formal publication through the relevant DOI), provides a link to the license, indicates if changes were made and the licensor is not https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 5/6 10/10/2015 RightsLink Printable License represented as endorsing the use made of the work. The full details of the license are available at http://creativecommons.org/licenses/by/4.0. CC BY NC SA: The CC BYNCSA license allows users to copy, to create extracts, abstracts and new works from the Article, to alter and revise the Article, provided this is not done for commercial purposes, and that the user gives appropriate credit (with a link to the formal publication through the relevant DOI), provides a link to the license, indicates if changes were made and the licensor is not represented as endorsing the use made of the work. Further, any new works must be made available on the same conditions. The full details of the license are available at http://creativecommons.org/licenses/byncsa/4.0. CC BY NC ND: The CC BYNCND license allows users to copy and distribute the Article, provided this is not done for commercial purposes and further does not permit distribution of the Article if it is changed or edited in any way, and provided the user gives appropriate credit (with a link to the formal publication through the relevant DOI), provides a link to the license, and that the licensor is not represented as endorsing the use made of the work. The full details of the license are available at http://creativecommons.org/licenses/byncnd/4.0. Any commercial reuse of Open Access articles published with a CC BY NC SA or CC BY NC ND license requires permission from Elsevier and will be subject to a fee. Commercial reuse includes:

 Associating advertising with the full text of the Article  Charging fees for document delivery or access  Article aggregation  Systematic distribution via email lists or share buttons

Posting or linking by commercial companies for use by customers of those companies.

20. Other Conditions:

v1.8

Questions? [email protected] or +18552393415 (toll free in the US) or +19786462777.

https://s100.copyright.com/App/PrintableLicenseFrame.jsp?publisherID=70&publisherName=ELS&publication=00145793&publicationID=11747&rightID=1&ty… 6/6 11/19/2015 RE: Letter of Permission (JBC Feedback Form) Scott Farrow ﴿RE: Letter of Permission ﴾JBC Feedback Form

Wed 9/23/2015 12:26 PM

Hi Dr. Farrow,

According to the JBC copyright permissions page: ﴾http://www.jbc.org/site/misc/Copyright_Permission.xhtml﴿ you have permission to use your article for your thesis.

If you have any additional questions please let me know.

Regards, Ed Marklin

These guidelines apply to the reuse of articles, figures, charts and photos in the Journal of Biological Chemistry, Molecular & Cellular Proteomics and the Journal of Lipid Research.

For authors reusing their own material:

Authors need NOT contact the journal to obtain rights to reuse their own material. They are automatically granted permission to do the following: • Reuse the article in print collections of their own writing. • Present a work orally in its entirety. • Use an article in a thesis and/or dissertation. Reproduce an article for use in the author's courses. ﴾If the author is employed by an academic institution, that • ﴿.institution also may reproduce the article for teaching purposes • Reuse a figure, photo and/or table in future commercial and noncommercial works. • Post a copy of the paper in PDF that you submitted via BenchPress. ◦ Only authors who published their papers under the "Author's Choice" option may post the final edited PDFs created by the publisher to their own/departmental/university Web sites. ◦ All authors may link to the journal site containing the final edited PDFs created by the publisher.

Please note that authors must include the following citation when using material that appeared in an ASBMB journal:

This research was originally published in Journal Name. Author﴾s﴿. Title. Journal Name. Year; Vol:pp‐pp. © the American" Society for Biochemistry and Molecular Biology."

For other parties using material for noncommercial use:

Other parties are welcome to copy, distribute, transmit and adapt the work — at no cost and without permission — for noncommercial use as long as they attribute the work to the original source using the citation above.

https://outlook.office.com/owa/#viewmodel=ReadMessageItem&ItemID=AAMkADFkYmZmZDAxLWM3ZmEtNGI3ZS1hMjdjLTkzNDJhZTFmODFkMQBGA… 1/2 11/19/2015 RE: Letter of Permission (JBC Feedback Form) Scott Farrow Examples of noncommercial use include: • Reproducing a figure for educational purposes, such as schoolwork or lecture presentations, with attribution. • Appending a reprinted article to a Ph.D. dissertation, with attribution.

For other parties using material for commercial use:

request permissions diagram

Navigate to the article of interest and click the "Request Permissions" button on the middle navigation bar. It will walk you through the steps for obtaining permission for reuse.

Examples of commercial use by parties other than authors include: • Reproducing a figure in a book published by a commercial publisher. • Reproducing a figure in a journal article published by a commercial publisher.

‐‐‐‐‐Original Message‐‐‐‐‐

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Comments sent via JBC Feedback Page ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ COMMENTS: .Regarding the below article:J Biol Chem. 2013 Oct 4;288﴾40﴿:28997‐9012 doi: 10.1074/jbc.M113.488585.

Dioxygenases catalyze O‐demethylation and O,O‐demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.

I am the author of the article above and would like to include it in my thesis. Our institution requires a letter from the publisher granting permission to include the published article in my final PhD thesis. Can someone please write a letter and e‐mail it to me at their earliest convenience. In the past, Sarah Crespi did this for a colleague. A similar letter would be greatly appreciated.

Kind Regards, Scott Farrow

https://outlook.office.com/owa/#viewmodel=ReadMessageItem&ItemID=AAMkADFkYmZmZDAxLWM3ZmEtNGI3ZS1hMjdjLTkzNDJhZTFmODFkMQBGA… 2/2 11/19/2015 Rightslink® by Copyright Clearance Center

Title: Stereochemical inversion of (S) reticuline by a cytochrome P450 If you're a copyright.com fusion in opium poppy user, you can login to Author: Scott C Farrow, Jillian M Hagel, RightsLink using your copyright.com credentials. Guillaume A W Beaudoin, Darcy Already a RightsLink user or C Burns, Peter J Facchini want to learn more? Publication: Nature Chemical Biology Publisher: Nature Publishing Group Date: Jul 1, 2015 Copyright © 2015, Rights Managed by Nature Publishing Group

Author Request

If you are the author of this content (or his/her designated agent) please read the following. If you are not the author of this content, please click the Back button and select an alternative Requestor Type to obtain a quick price or to place an order.

Ownership of copyright in the article remains with the Authors, and provided that, when reproducing the Contribution or extracts from it, the Authors acknowledge first and reference publication in the Journal, the Authors retain the following nonexclusive rights:

a) To reproduce the Contribution in whole or in part in any printed volume (book or thesis) of which they are the author(s).

b) They and any academic institution where they work at the time may reproduce the Contribution for the purpose of course teaching.

c) To reuse figures or tables created by them and contained in the Contribution in other works created by them.

d) To post a copy of the Contribution as accepted for publication after peer review (in Word or Text format) on the Author's own web site, or the Author's institutional repository, or the Author's funding body's archive, six months after publication of the printed or online edition of the Journal, provided that they also link to the Journal article on NPG's web site (eg through the DOI).

NPG encourages the selfarchiving of the accepted version of your manuscript in your funding agency's or institution's repository, six months after publication. This policy complements the recently announced policies of the US National Institutes of Health, Wellcome Trust and other research funding bodies around the world. NPG recognises the efforts of funding bodies to increase access to the research they fund, and we strongly encourage authors to participate in such efforts.

Authors wishing to use the published version of their article for promotional use or on a web site must request in the normal way.

If you require further assistance please read NPG's online author reuse guidelines.

For full paper portion: Authors of original research papers published by NPG are encouraged to submit the author's version of the accepted, peerreviewed manuscript to their relevant funding body's archive, for release six months after publication. In addition, authors are encouraged to archive their version of the manuscript in their institution's repositories (as well as their personal Web sites), also six months after original publication.

v2.0

https://s100.copyright.com/AppDispatchServlet#formTop 1/2 11/19/2015 Rightslink® by Copyright Clearance Center

https://s100.copyright.com/AppDispatchServlet#formTop 2/2

PERMISSION TO USE COPYRIGHTED MATERIAL IN THESIS

REQUEST I, Scott C. Farrow, am a PhD. Candidate at the University of Calgary and am preparing my final thesis. I am requesting permission to include materials from the co-authored publication of which you are one of the co-authors as described below. The source(s) of included materials will be fully identified in my thesis. The thesis will be digitized and available online through the Library of the University of Calgary, and Library and Archives Canada.

Title of thesis: Expanding the Role of Oxidoreductases in Benzylisoquinoline Alkaloid Metabolism in Opium poppy. Degree: Doctor of Philosophy Graduating Year: 2015

Author of thesis: Scott Cameron Farrow

PERMISSION Permission is hereby granted to Scott C. Farrow, the Library of the University of Calgary, and the Library and Archives Canada to reproduce the following publications in the aforementioned thesis.

Title: Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

Co-author of publication: Dr. Jillian Hagel