Enzyme Structure, Function, and Evolution in Flavonoid Biosynthesis by Geoffrey Liou B.A. Molecular and Cell Biology University of California, Berkeley, 2013 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2019 © 2019 Massachusetts Institute of Technology. All rights reserved. Signature of Author _____________________________________________________________ Geoffrey Liou Department of Biology May 24, 2019 Certified by ___________________________________________________________________ Jing-Ke Weng Assistant Professor of Biology Thesis Supervisor Accepted by __________________________________________________________________ Amy E. Keating Professor of Biology Co-Director, Biology Graduate Committee 1 2 Enzyme Structure, Function, and Evolution in Flavonoid Biosynthesis by Geoffrey Liou Submitted to the Department of Biology on May 24, 2019 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Abstract Plant specialized metabolism is a key evolutionary adaptation that has enabled plants to migrate from water onto land and subsequently spread throughout terrestrial environments. Flavonoids are one particularly important class of plant specialized metabolites, playing a wide variety of roles in plant physiology including UV protection, pigmentation, and defense against herbivores and pathogens. Flavonoid diversity has increased in conjunction with land plant evolution over the past 470 million years. This dissertation examines the structure, function, and evolution of enzymes in the flavonoid biosynthetic pathway. First, we structurally and biochemically characterized orthologs of chalcone synthase (CHS), the enzyme that catalyzes the first step of flavonoid biosynthesis, from diverse plant lineages. By doing so, we gained insight into the sequence changes that gave rise to increased reactivity of the catalytic cysteine residue in CHS orthologs in euphyllophytes compared to basal land plants. We then developed methods and transgenic plant lines to study the in vivo function of these CHS orthologs, as well as whether their functional differences play a ​ ​ role in redox-based regulation of flavonoid biosynthesis. Finally, we examined enzymes involved in the biosynthesis of galloylated catechins, a highly enriched class of flavonoids in tea that are thought to have health benefits in humans. These findings contribute to an understanding of the evolution of enzyme structure and function in flavonoid biosynthesis, and how it has facilitated the adaptation of plants to a wide variety of terrestrial habitats. Thesis Supervisor: Jing-Ke Weng Title: Assistant Professor of Biology 3 4 Acknowledgements First and foremost, I would like to thank my thesis advisor, Jing-Ke Weng. I feel very fortunate to have joined the lab as the first group of students and to have watched it grow over the years. I remember being instantly captivated by the research when I saw your presentation to the first-year graduate students. It was a perfect alignment of my interests at the time, structural biology and plant biology, but my experience has turned out to be more than I could ever have imagined. Your enthusiasm for and breadth of knowledge in all aspects of biology has been inspiring and has taught me to keep learning and discover my passions. Your insight and support has been invaluable in helping me think more broadly and work past the difficult parts of my research. To the members of the Weng Lab, I can’t imagine a better group of people to work with. Tim Fallon, you have been a great classmate, seat neighbor, labmate, roommate, and most of all friend throughout the years. I’ll never forget the first time I saw fireflies when we went to New Jersey to collect Photinus pyralis. Olesya Levsh, I’m so glad to have joined the lab together with ​ ​ you. Your kindness set the tone of the lab from the beginning and has left a mark in the form of many lab traditions. Joe Jacobowitz, thanks for all the fun game nights and putting up with our teasing. Sophia Xu, it has been great sharing Asian snacks and bonding as baymates despite our differences (Go Bears!). Bena Chan, thank you for warmly welcoming me to the lab during my rotation, and for staying in touch and all your help in your current position in the Metabolomics Core. Valentina Carballo, thank you for everything you do to keep the lab running and making everyone feel like part of a family. Fu-Shuang Li, it has been inspiring to see your dedication to your family and your work. Mike Spence, thanks for imparting your wisdom in biochemistry and life experience over the years. Bastien Christ, thank you for sharing your knowledge of plants, fondue, and sense of adventure. Tomáš Pluskal, I have learned so much about metabolomics, mind-bending films, and life from you, and I aspire to be an international man of mystery like you. Roland Kersten, thanks for your help over the years and bringing some California spirit to the lab. Chengchao Xu, Andrew Mitchell, Yasmin Chau, Matthew Hill, Chris Glinkerman, Menglong Xu, Anastassia Bobokalonova, Amy Zhang, Sheena Vazquez, Brian Levine, Jack Liu, Michael Gutierrez, Naoki Wada, Colin Kim, and others: thank you for making this lab a fun place to work. It has been a pleasure and privilege to get to know such an interesting, diverse group of people. Thanks to Biograd 2013 for all the memories. In particular, Chetan, Aneesha, Rachit, Emir, Nicole, and Amelie, it’s been great sharing our journey through grad school and getting together to relax and unwind. Thank you to my friends at MIT Japanese Lunch Table, in the Cal Alumni Club of New England, in the Boston area, in New York, back home in California, and in Japan, and also my extended family in Taiwan, for all the fun times and keeping me connected to the things I love outside of school. Special thanks to MIT Japan Program Director Chris Pilcavage, Miyuki-san, Masako-san, the Baber family, Joey, Kristine, Matthew, Mark, Diana, Sherman, Roger, Yuzo, Heechan, Willie, Sam, David, Alex, Daniel, Tomoya, Moka, Chihhi, Yuihan, and Dahyun, among many others. 5 Finally, I want to thank my family. Kerry and Zachary, it’s been great to watch you follow your own paths as we become independent adults. To my mother and father, thank you for everything you have done for us, first and foremost valuing our education, which has brought us to where we are today. We are very fortunate to have so much opportunity here in the United States, and I am grateful for all your sacrifice and hard work that has brought us here. 6 Table of Contents Abstract 3 Acknowledgements 5 Table of Contents 7 Chapter 1. Introduction 9 ​ ​ Overview of land plant evolution 9 Plant specialized metabolism 11 ​ Flavonoid biosynthesis and diversity 13 ​ Type III polyketide synthases 18 Applications of plant metabolic and enzyme engineering 21 ​ Concluding remarks 24 ​ References 26 ​ Chapter 2. Mechanistic basis for the evolution of chalcone synthase catalytic cysteine ​ ​ reactivity in land plants 31 ​ Abstract 32 ​ Introduction 33 ​ Results 37 ​ Basal-plant CHSs contain reduced catalytic cysteine in their crystal structures 37 ​ Basal-plant CHSs only partially complement the Arabidopsis CHS-null mutant 41 ​ The pKa of the catalytic cysteine is higher in basal-plant CHSs than in euphyllophyte ​ ​ ​ CHSs 42 ​ Residues near the active-site cavity affect the pKa and reactivity of the catalytic ​ ​ ​ cysteine 43 ​ Molecular dynamics simulations reveal differences in active-site interactions between basal-plant and euphyllophyte CHSs 49 Discussion 54 ​ Materials and Methods 58 References 65 ​ Supporting Information 68 Chapter 3. Regulation of chalcone synthase activity in vivo by oxidation of the catalytic ​ ​ cysteine 81 ​ Abstract 82 ​ Introduction 83 ​ Results 87 ​ tt5 mutant Arabidopsis thaliana accumulates naringenin and can be used for metabolic ​ ​ ​ tracing to measure CHS activity in vivo 87 ​ tt5 mutant Arabidopsis thaliana accumulates both enantiomers of naringenin 89 ​ ​ ​ Generation of and metabolic tracing with tt5 and mbs1-1 mutant Arabidopsis crosses 89 ​ ​ ​ ​ 7 The catalytic cysteine in AtCHS is more sensitive to in vitro oxidation than in SmCHS 91 ​ FLAG-tag purification and western blotting of CHS 95 ​ Generation and characterization of transgenic Arabidopsis thaliana lines expressing ​ ​ FLAG-tagged CHS orthologs 98 ​ Discussion and Future Directions 101 ​ Materials and Methods 103 ​ References 111 ​ Appendix. Investigation of galloylated catechin biosynthetic enzymes in tea 113 ​ ​ ​ Abstract 114 ​ Introduction 115 ​ Results 119 ​ CsUGGT expression in Nicotiana benthamiana produces β-glucogallin 119 ​ ​ ​ Identification of ECGT candidate genes 119 ​ Nicotiana benthamiana leaf protein extraction fails to show ECGT activity 121 ​ ​ Discussion and Future Directions 124 ​ Materials and Methods 126 ​ References 131 ​ 8 Chapter 1 Introduction Overview of land plant evolution Terrestrial life as it exists today was seeded and shaped by the transition of plants from water to land. Embryophytes, or land plants, evolved from the charophycean green algae approximately 470 million years ago (Kenrick & Crane, 1997). Life on land brought numerous ​ ​ challenges to plants previously ameliorated by an aquatic environment: ultraviolet radiation, desiccation, lack of structural support, and gas exchange. As other clades of life also adapted to land, plants also needed to fend off pathogens and herbivores.