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Structural Studies of Rebeccamycin, Staurosporine, and Violacein Biosynthetic Enzymes

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Structural Studies of Rebeccamycin, Staurosporine, and Violacein Biosynthetic Enzymes Structural studies of rebeccamycin, staurosporine, and violacein biosynthetic enzymes by Katherine Snoda Ryan B.S. Biological Chemistry The University of Chicago, 2002 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOPHYSICAL CHEMISTRY AND MOLECULAR STRUCTURE AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY JULY 2008 © Katherine Snoda Ryan. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publically paper and electronic copies of this thesis document in whole or in part. Signature of Author:_______________________________________________________ Department of Biology July 3, 2008 Certified by: _____________________________________________________________ Catherine L. Drennan Professor of Chemistry and Biology Thesis Supervisor Accepted by: ____________________________________________________________ Steven P. Bell Professor of Biology Chair, Biology Graduate Committee This doctoral thesis has been examined by a Committee of the Department of Biology as follows: Professor Amy E. Keating __________________________________________________ Committee Chairman Professor Catherine L. Drennan ______________________________________________ Research Supervisor Professor Thomas U. Schwartz ______________________________________________ Committee Member Professor JoAnne Stubbe ___________________________________________________ Committee Member Professor Sean J. Elliott ____________________________________________________ Committee Member Boston University 2 Structural studies of rebeccamycin, staurosporine, and violacein biosynthetic enzymes by Katherine Snoda Ryan Submitted to the Department of Biology on July 3, 2008 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biophysical Chemistry and Molecular Structure ABSTRACT The biosynthesis of medically relevant bisindole natural products rebeccamycin, staurosporine, and violacein from the common starting material L-tryptophan involves shared enzymatic transformations. However, the pathways diverge at two steps, each involving a reactive, bisindole intermediate. We have taken a structural approach to characterize the biosynthetic enzymes responsible for these divergence points in each pathway. One major difference between rebeccamycin and staurosporine is the oxidative state of the C-7 carbon. The enzymes RebC and StaC (65% sequence identity) control the oxidative outcome at the C-7 position. Our work on the rebeccamycin biosynthetic enzyme RebC has enabled us to crystallographically ‘trap’ its putative substrate and to identify a likely enzymatic function for RebC in controlling the outcome of this key step in rebeccamycin biosynthesis. We have also used the structure of RebC with reduced flavin to probe the likely reaction cycle of a single round of flavin-based hydroxylation chemistry in RebC. Finally, the structure of RebC has allowed us to use a structure-based mutagenesis approach to install a higher affinity binding site for FAD in the RebC homologue StaC, which normally binds FAD weakly. The resulting protein possesses RebC-like properties. Another divergence point between these biosynthetic pathways is the presence or absence of the VioE enzyme, which diverts a reactive intermediate toward violacein precursor. Our structural studies on VioE have shown that this enzyme shares a fold with lipoprotein carrier proteins. A series of site-directed mutagenesis experiments on residues around a PEG molecule bound in the VioE structure have revealed the likely location of the active site in VioE. 3 Collectively, our work has uncovered a key theme in bisindole enzymology: RebC, StaC, and VioE function by intercepting reactive substrate molecules, stabilizing these molecules in their active sites, and promoting their conversion to on-pathway products. In the absence of these enzymes, the reactive substrate molecules decompose to shunt products. RebC, StaC, and VioE therefore function in part simply as ‘stabilizing’ enzymes, providing enough chemical energy to prevent the production of spontaneously derived shunt products. Thesis Supervisor: Catherine L. Drennan Title: Professor of Chemistry and Biology and HHMI Investigator 4 To Mom, Dad, Robert, and Hong 5 Acknowledgements Thank you to Cathy Drennan, who took a chance on me and guided me through my Ph.D. She taught me how to solve structures and write papers, she offered me mentorship on all topics at all hours, she rooted for me at seminars, she worried about my future, and she inspired me to become a crystallographer. I could not have finished my degree or considered a scientific career without her. Thank you to my collaborator, Christopher T. Walsh, for his insight over the course of these investigations and for teaching me so much about the practical aspects of carrying out research. He has a magical ability to see majestic problems where others see simple biochemical pathways, and I also thank him for graciously giving his advice about future directions to pursue in science. Thank you to Angie Belcher, my first advisor, who saw that our differences were between the realms of science and engineering, and who allowed me to stay in her lab, as a friend and as a mentor, until I found the right path. My collaborators – Annaleise Howard-Jones, Carl Balibar, and Mike Hamill – have been great scientists from across the river, and our interactions made these projects far more ambitious than I could have hoped for alone. My committee members, Amy Keating and Thomas Schwartz, have been a tremendous source of support to me. Amy spoke honestly to me and helped me to be more critical of my work. Thomas helped me think from different angles about crystallography, and even went with me on a trip to the Advanced Photon Source. A multitude of other professors have helped me through my graduate education. Dave Ballou welcomed me to his lab and to his home, and introduced me to the world of stopped-flow spectroscopy. His enthusiasm for life and energy for science are enviable. Sean Elliott, a great scientist and funny guy, has helped me with my work through extremely helpful discussions and in electrochemical work. Deborah Zamble has collaborated with me over a series of interesting projects, has given me wonderful advice, and welcomed me into her laboratory in Toronto. JoAnne Stubbe, the smartest enzymologist in the world, took the time to help me think through mechanisms and talk with me about my plans. I also want to thank a number of faculty members who have helped me through interactions, formal and informal, that have guided and inspired me: Mohammed Movassaghi, Stuart Licht, Barbara Imperiali, Sarah O’Connor, Collin Stultz, Alan Grossman, Phil Lessard, and Ed Grant. I also want to thank the professors who encouraged me to go to graduate school at all: Jocelyn Malamy, Laurie Mets, and Daphne Preuss. Additionally, I want to thank Becky Stark, who was my first collaborator, back in college, and my host for adventures in Bryce Canyon. My labmates in graduate school have all been friends as well as colleagues. Julie Norville, a- friendly-but-ready-to-rock-the-world-of-science type, has always been an inspiration. Hector Hernandez doled out help before he was even my labmate and always made me laugh. Lisen Kaiser, while not in my lab, still kindly trained me to use an FPLC and taught me all about Swedish fashion (I’m still a hopeless case, however). Eric Krauland and Saeeda Jaffar were the best distractions one could ask for. Paul Hubbard was a wonderful teacher and good friend. Christine Phillips helped me out before she knew me and again and again after she did. Ainsley Davis I thank for his quiet insights and unending kindness. Steve Kottmast was a great friend when times were tough. Chiang and Ki Tae Nam always kept me laughing. Leah Blasiak always 6 took the time to help me strategize and think about my research problems. Yan Kung was a constant source for interesting ideas and thoughtful commentary. Jess Vey’s quiet tenacity was an inspiration. Danny Yun, who always prevented me from working with endless, entertaining stories, was also kind enough to try to teach me some molecular biology. Cintyu Wong never ceases to impress me, and counts in my mind as my bravest co-worker. Kaitlyn Turo, who was only a freshman in college when I met her, is a superstar undergraduate research assistant, a fashion consultant, and entertaining to boot. Thank you as well to many other people in the lab, and down the hall, too: Dan, Kiley, Sreekar, Brian, Andrew, Lee, Asher, Amy, Ramon, Jun, Bill, Christina, Laura P., Peter, Melva, Laura J., Mary, Wan-Chen, Mathew, and Eugene. To other wonderful friends. First, Chris Rycroft, who made me decide the British were alright, and for being one of my closest friends. To Vivian Lei, future marathon champion and the owner of the most welcoming heart ever made. To Caterina Schweidenback, who kept me well-fed and laughing. To Tony Lau, who is hilarious and thoughtful. To Shan Wu and Valentina Urbanek, for great times over brunch and in Acadia. To fantastic roommates – Yujia Xia, Joey Weiner, Will Hafer, Kris Kopanski, Jessica Brown, Ramya Rajagopolan, Amanda Miller, and Katy Thorn. To the rest of the crew: Tyrone Hill, Shawn Kuo, Song-Hee Paik, Steve Kohen, Natalija Jovanovich, Mitch Peabody, Bruce Wu, Vikas Anant, Karen Lee, Joe Rumpler, Yuhua Hu, and Vickram Mangalgiri. To my biology classmates, each of them, and especially Jessica Brown, Sarah Bissonette, Duaa Mohammed,
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