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Structure and Biochemistry of Cytochromes P450 Involved in the Biosynthesis of Macrolide Antibiotics

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Structure and Biochemistry of Cytochromes P450 Involved in the Biosynthesis of Macrolide Antibiotics Structure and Biochemistry of Cytochromes P450 Involved in the Biosynthesis of Macrolide Antibiotics by Matthew D. DeMars II A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2017 Doctoral Committee: Professor David H. Sherman, Chair Professor David P. Ballou Professor John Montgomery Professor Stephen W. Ragsdale Professor Janet L. Smith Matthew D. DeMars II [email protected] ORCID iD: 0000-0002-7268-5286 © Matthew D. DeMars II 2017 Dedication This work is dedicated to the memory of my father, Matthew David DeMars. ii Acknowledgments I have been exceedingly fortunate to have worked with some exemplary scientists during the formative years of my scientific career, and it would simply be impossible to express the full depth and breadth of my gratitude in the rather limiting context of this acknowledgments section. Quite fortuitously, I joined the laboratory of Dr. Rudi Fasan as an undergraduate student at the University of Rochester. Although I had limited experience in carrying out research at the interface of chemistry and biology, Dr. Fasan recognized my potential as well as genuine interest in the project he had assigned me and provided me with a considerable degree of autonomy shortly after I joined his lab. The work that I carried out as an undergraduate helped to solidify my fundamental affinity for interdisciplinary research, providing me with the drive to apply to and matriculate at the University of Michigan in pursuit of an advanced degree in chemical biology. As I look back five years, it is surprising that Dr. David Sherman was barely even on my radar of potential research advisors when I was considering labs in which to rotate. However, my decision to come to Michigan and ultimately to join his lab is one that I will never regret. The opportunity to perform cutting-edge and exciting research in a highly cooperative environment with some of the most skilled and intelligent people I have ever known has been a true blessing, and I could not have asked for a better lab in which to spend my tenure as a graduate student. First and foremost, I would like to express my utmost gratitude to Dr. Sherman for giving me the freedom to pursue my own research interests and for his unabated support over the past four years. His hands-off mentoring style was appropriately complemented by his approachability and unremitting willingness to discuss and provide constructive feedback on a variety of matters whether or not they were directly related to research. Ultimately, my experience in his lab fostered my development into an independent scientist with the confidence to ask and attempt to answer my own iii research questions. I also owe a debt of gratitude to my committee members: Dr. David Ballou, Dr. John Montgomery, Dr. Stephen Ragsdale, and Dr. Janet Smith. Their thoughtful questions, constructive criticisms, and valuable insights challenged me to think outside of the box and were truly instrumental in my development into a more mature researcher. One of the most rewarding aspects of working in the Sherman lab was the opportunity to be a part of several interdisciplinary collaborations with labs across the country as well as overseas. In particular, I am extremely grateful to have gotten the chance to work with Dr. Larissa Podust, whose prolific career studying cytochromes P450 has made her a bona fide expert in the structure and biochemistry of these enzymes. Her work in solving the structures of two P450s that I extensively describe herein served as a critical foundation for many of the biochemical experiments that I later designed and carried out. More recently, we have established a collaboration with Dr. Kendall Houk, whose expertise in computational chemistry has provided an invaluable resource for better understanding dynamic processes during enzyme catalysis. I very much look forward to continuing our work with him and his lab (especially graduate student Song Yang) over the next year. Finally, I would like to thank Dr. John Montgomery and Dr. Pavel Nagorny for providing me with the opportunity to work with some of their students (Michael Gilbert, Jessica Stachowski, and Jia Hui Tay) on collaborative projects that are not described in the present body of work. My coworkers in the Sherman lab have been and continue to be a constant source of inspiration. I have truly never met a more motivated, intelligent, and talented group of people, and it has been an honor to work alongside them for the past four years. I would especially like to thank past members Dr. Alison Narayan and Dr. Sungryeol Park as well as current members Dr. Andrew Lowell, Dr. Sean Newmister, and Dr. Ashu Tripathi for their expert advice in many areas of personal scientific ignorance. I also want to thank my bay-mates Vik Shende, Amy Fraley, and Rosa Vasquez for putting up with my antics on a daily basis. I am indebted to them along with all other members of the lab for helping to take the edge off of graduate school. Finally, I would like to extend my gratitude to Pam Schultz for being irreplaceable in her role as iv lab manager as well as former and current lab secretaries Shamilya Williams and Debbie Lounds, respectively, for all of their amazing work in helping to maintain a functional and jovial work environment. I would also like to take this opportunity to thank the Program in Chemical Biology (PCB) for admitting me as a graduate student and for being an unwavering source of support during my time here at Michigan. Program director Dr. Anna Mapp and administrators Laura Howe and Traci Swan worked hard to ensure that the entire graduate school process went as smoothly as possible for me and many others, and I sincerely thank them for all of their efforts in this respect. I feel especially fortunate to have been one of ten fantastic students that matriculated at Michigan during the 2012- 2013 academic year. The camaraderie we shared is something I will forever remember and be grateful for. During my second and third years as a graduate student at Michigan, I was fortunate enough to receive funding through the Cellular Biotechnology Training Program, which provided me with enhanced education in different areas of biotechnology. As per one of its requirements, I was able to spend Summer 2014 in South Korea working under Dr. Eung-Soo Kim in his “Microbial Molecular Biotechnology Laboratory” at Inha University. I am grateful to Dr. Sherman for presenting me with this option, to Dr. Kim for hosting me in his laboratory, and to the Center for Selective C–H Functionalization (CCHF) for funding my entire trip. As my final year in the PCB was funded by the Rackham Predoctoral Fellowship, I would also like to thank the Rackham Graduate School for its financial support. One of the reasons I decided to come to Michigan for graduate school was the prospect of being able to simultaneously pursue my passion for early classical music. Indeed, I was afforded the opportunity to enroll in continuo and Baroque chamber ensemble courses at the School of Music starting from my very first semester as a graduate student. I want to thank Profs. Edward Parmentier and Joseph Gascho for providing me with the chance to learn about and play amazing music in a world-class university setting. Serendipitously, I also met another graduate student (Wallace Chan) whose passion for early music closely rivals mine. It has been a privilege to play music v with him and some of our mutual friends on a regular basis, and I have enjoyed going with him and his wife Mia to all of the early music concerts Ann Arbor offers every year. Last, but certainly not least, I would like to express my deepest gratitude to my family. My parents, Matt (who just recently passed away) and Farolyn DeMars, have been there for me from the beginning and have played key roles in fostering my persistent desire to learn and create. Although they are truly unique individuals with different sets of interests and beliefs, they have always emphasized their mutual love for me and have both sacrificed so much for me to be where I am today. I’m notoriously not a very emotional person, so my gratitude and love are not always explicitly expressed, but I would like my parents to know that these feelings are always there. I also thank my stepparents, Michael Reid and Denise DeMars, for all the love and support they have given me over the years. My godparents, Larry and Jan Singer, have always taken a keen interest in my life, and I thank them for their constant love, kindness, and generosity. My grandparents, Dave and Carol DeMars, kindly hosted me in their home for several weeks every summer when I was a child/young teenager. Looking back, I cannot think of anything more they could have done to demonstrate their love for me. I would also like to thank them for providing me with the chance to take a break from writing this dissertation by joining them and the rest of the family on an Alaskan cruise this past summer. Finally, I thank my late grandfather, Jack Weinberg, who passed away just as I was starting as an undergraduate at the University of Rochester back in Fall 2008. He was someone I truly looked up to as a role model for how to treat others with kindness, warmth, and generosity, and I will forever cherish the memories I have of spending time with him. vi Table of Contents Dedication .......................................................................................................................
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