People of mediocre ability sometimes achieve outstanding success because they don’t know when to quit. Most men succeed because they are determined to. -George Allen, Sr., ii The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids Dylan Edward Ryan Levac, B.Sc. Biotechnology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Biological Sciences, Brock University St. Catharines, Ontario ©2013 iii ABSTRACT The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti- neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, iv Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity. v ACKNOWLEDGEMENTS First and foremost, I wish to express my appreciation to those who have contributed directly the planning and execution of this work. I am extremely grateful to Professor Vincenzo De Luca. His guidance and helpful discussions, especially during the difficult stages, has been critical to my success. Dr. De Luca has guided me through a maturation process, both as a professional scientist and as a person. I am indebted for his understanding, compassion and support during tough times, especially a difficult 2012. I would like to also thank the rest of my thesis committee: Prof. Charles Després and Prof. Costa Metallinos for their encouragement, and insightful comments. I begin my preparations for every experiment, or every presentation, with considering a very specific question; ‘what criticism will Charles ask?’ This extra pressure has given me an additional push to develop as a scientist and public speaker. This thesis would not have been possible had it not been for the efforts of two key individuals. I would like to thank above all Dr. Jun Murata. Although Jun left before my Ph.D. began, he has been central to my professional development, molding me into the scientist I am today. Had it not been for his seemingly endless desire to help and mentor me I do not believe I would be here to defend this thesis. I would also like to thank Dr. Fang Yu. Fang has been key to my further maturation as a scientist. Where Jun would imagine elegant approaches to solving problems, Fang has taught me brute force, persistence, and a seemingly endless well of optimism. I apply these opposing philosophies not only my research, but also in my personal life. I cannot thank you both enough. vi I would also like to thank past and present lab members and friends at Brock; Brent Wiens, Dr. Kyung-Hee Kim, Dr. Ashok Gosh, Dr. Sayaka Masada Atsumi, Vonny Salim, Antje Tham, Jonny Roepke, Dr. Dawn Hall, Dr. David Crandles, Dr. Pat Boyle, Dr. Hannes Lesch, Dr. Rob Caroll, Dr. Kevin Finn, Dr. Josh Zaifman. Furthermore I would like to thank the Brock University administration, science stores and custodial staff. We normally take for granted all the work they do that allows us to focus our attention on research and our studies. I would like to especially thank Nick Mef. You were always there and you always had time to talk. I will deeply miss our conversations. I would like to thank especially Raul Masseur. I had the great pleasure of meeting Raul in 2007 when I was searching for opportunities to experience other cultures. Raul invited me to participate in Solidarity Experiences Abroad that year, traveling in Peru. That experience impacted my world view so much, and so positively, that I participated again in 2009, traveling this time to Brazil. I cannot imagine becoming the person I am today without these two key, life changing, opportunities and the many deeply insightful conversations I have had with Raul throughout the years. Thank you very much my friend. I will do my best, but words cannot express the gratitude I have for my parents, family, and very close friends. I would not be here if it not for all of you. Special thanks are due to my mother, Deborah, and father, Jerome. Your endless support has been central to my ability to succeed. I can only hope that I will transition my academic success in to similar professional success. I promise to achieve that success on a much vii more reasonable and shorter time frame. One cannot be an official student for forever and I am sure you are sighing in relief that I am finally moving to the work force. Finally, I would like to acknowledge the most important person in my life – my fiancée, Brandi. She has been a constant source of strength and support. I would have never completed this program without Brandi and I look forward to spending the rest of our lives together. viii TABLE OF CONTENTS ABSTRACT......................................................................................................................iii ACKNOWLEDGEMENTS............................................................................................. v TABLE OF CONTENTS ...............................................................................................vii LIST OF TABLES......................................................................................................... xiv LIST OF FIGURES ....................................................................................................... xv LIST OF ABBREVIATIONS ..................................................................................... xvii CHAPTER 1 – INTRODUCTION.................................................................................. 1 1.1 THESIS OUTLINE........................................................................................... 3 1.2 BIBLIOGRAPHY............................................................................................. 6 CHAPTER 2 – LITERATURE REVIEW - SPECIALIZED METABOLISM AND SILENT BIOCHEMICAL PATHWAYS ...................................................................... 8 2.1 LIFE NECESSITATES SPECIALIZED ADAPTATIONS ............................ 8 2.2 EVOLUTIONARY EXPANSION OF CHEMICAL STRUCTURES FOR DIVERSE BIOLOGICAL FUNCTION ....................................................... 9 2.3 THE COST OF SPECIALIZED METABOLISM ......................................... 12 2.4 BALANCING ALLOCATION COSTS ........................................................ 13 2.4.1 THE CARBON-NITROGEN BALANCE THEORY .................... 15 2.4.2 THE GROWTH-DEVELOPMENT BALANCE THEORY .......... 16 2.4.3 THE RESOURCE AVAILABILITY THEORY ............................ 17 2.4.4 EVOLVED COST BALANCE;CYANOGENIC GLUCOSIDES.. 19 2.5 ABERRANT METABOLISM VERSES THE CHEMICAL ARMS RACE ................................................................................................................... 21 2.6 THE SCREENING HYPOTHESIS ............................................................... 24 2.7 SILENT METABOLIC PATHWAYS IN PLANTS ..................................... 25 ix 2.8 BIOPROSPECTING FOR NOVEL TOCOPHEROL-LIKE N- METHYLTRANSFERASES ............................................................................... 29 2.9 BIBLOIGRAPHY .........................................................................................