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Carbonic Anhydrase and Epi-Isozizaene Synthase

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Carbonic Anhydrase and Epi-Isozizaene Synthase University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Spring 2010 Structure, Function and Applications of Metal-Requiring Enzymes: Carbonic Anhydrase and Epi-Isozizaene Synthase Julie A. Aaron University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Aaron, Julie A., "Structure, Function and Applications of Metal-Requiring Enzymes: Carbonic Anhydrase and Epi-Isozizaene Synthase" (2010). Publicly Accessible Penn Dissertations. 142. https://repository.upenn.edu/edissertations/142 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/142 For more information, please contact [email protected]. Structure, Function and Applications of Metal-Requiring Enzymes: Carbonic Anhydrase and Epi-Isozizaene Synthase Abstract Cryptophane Biosensors for Targeting Human Carbonic Anhydrase Cryptophanes represent an exciting class of xenon-encapsulating molecules that can be exploited as probes for nuclear magnetic resonance imaging. A series of carbonic anhydrase-targeting, xenon-binding cryptophane biosensors were designed and synthesized. Isothermal titration calorimery and surface plasmon resonance measurements confirmed nanomolar affinity between human carbonic anhydrase II and the cryptophane biosensors. A 1.70 Å resolution crystal structure of a cryptophane-derivatized benezenesulfonamide human carbonic anhydrase II complex was determined, and shows how an encapsulated xenon atom can be directed to a specific biological target. Furthermore, this work illustrates the utility and promise of developing xenon biosensors to diagnose human diseases characterized by the upregulation of specific carbonic anhydrase biomarkers, specifically human carbonic anhydrase IX and XII. Structural Studies of epi-Isozizaene Synthase from Streptomyces coelicolor The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 Å resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg2+ ions, inorganic pyrophosphate (PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 Å resolution. Comparison of these two structures provides the first view of conformational changes equirr ed for substrate binding and catalysis in a bacterial terpenoid cyclase, and enables a comparison of substrate recognition amongst terpenoid synthases from different domains of life. Mutagenesis of aromatic residues in the enzyme active site alters the cyclization template and results in the production of alternative sesquiterpene products. The structure and activity of several active site mutants have been explored. The 1.64 Å resolution crystal structure of F198A EIZS in a complex with three Mg2+ ions, PPi, and BTAC reveals an alternative binding orientation of BTAC, whereas the crystal structures of L72V, A236G and V329A EIZS reveal an unchanged BTAC binding orientation. Alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor David Christianson Keywords X-ray crystallography, terpene cyclase, enzyme catalysis, biosensor, xenon Subject Categories Biochemistry, Biophysics, and Structural Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/142 STRUCTURE, FUNCTION AND APPLICATIONS OF METAL-REQUIRING ENZYMES: CARBONIC ANHYDRASE AND EPI-ISOZIZAENE SYNTHASE JULIE ANNE AARON A Dissertation in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 ___________________________ Dr. David W. Christianson, Professor, Chemistry Supervisor of Dissertation ___________________________ Dr. Gary A. Molander , Professor, Chemistry Department Chairman Dissertation Committee Dr. Ronen Marmorstein, Professor, Chemistry and Wistar Institute Dr. Ivan J. Dmochowski, Associate Professor, Chemistry Dr. Barry S. Cooperman, Professor, Chemistry “One must still have chaos in oneself to be able to give birth to a dancing star.” - Nietzsche I dedicate this thesis to my family, for supporting me, and to Dr. Li and Mr. Doketis, for inspiring me. ii Acknowledgements My graduate experience has been more rewarding then I ever imagined. I am deeply indebted to my mentors, colleagues, friends, and family who have supported, encouraged, and celebrated with me these past 5 years. First and foremost, I would like to thank my dissertation advisor, Dr. David Christianson. David is an excellent mentor, both personally and professionally. I admire his dedication and enthusiasm for teaching, and am grateful for the opportunity to work in his lab. The balance of support and scientific independence with which he provided me was essential to my success. I would also like to extend my greatest appreciation to the members of my dissertation committee, Dr. Ronen Marmorstein, Dr. Ivan Dmochowski and Dr. Barry Cooperman for their advice and guidance. I would especially like to acknowledge Dr. Marmorstein, Director of the Chemistry-Biology Interface (CBI) Training Program, for his enthusiastic support of the CBI Program and Chemical Biophysics Mini-Symposia Series (CBMS). I have had the privilege of working in a lab filled with incredibly supportive and engaging colleagues, with whom I have enjoyed friendships, both inside and outside the lab. I have especially enjoyed our camaraderie during countless synchrotron trips, where despite the lack of sleep, we managed to have a lot of fun. I would like to especially thank Dr. Luigi Di Costanzo, and Dr. Heather Gennadios who served as my mentors when I first joined the lab, and Dr. Dan Dowling, Dr. Kathryn Cole and Kate Thorn for iii their friendship and great chats over coffee/tea over the years. I would also like to thank the other members of our lab, Dr. German Gomez, Dr. Hyunshun Shin, Dr. Kevin Jude, Dr. Sangeetha Vedula, Dr. Katya Shishova, Dr. Tatiana Zakharian, Dr. Monica Ilies, Dr. Mustafa Koksal Dr. Mo Chen, Patrick Lombardi and Cristina Virgilio for their friendship and advice over the years. The work I present herein is the result of two excellent collaborations. First, I would like to thank Dr. Aru Hill and Jenny Chambers of Dr. Dmochowski’s Lab at the University of Pennsylvania. Jenny Chambers is one of the most beautiful people I have ever met, inside and out, and Dr. Aru Hill, is the smartest graduate student I have had the privilege of knowing. I am very grateful to them for the success of the CAII-cryptophane project. Secondly, my thanks go to Dr. David Cane at Brown University. I am grateful for the opportunity to twice visit his labs in Providence, to perform experiments with Dr. Xin Lin and Dr. Wayne Chou on the ep i-isozizaene synthase project. I am indebted to members of the Departments of Chemistry, and Biochemistry & Biophysics Departments for their financial and administrative support of my extracurricular endeavors. The success of the CBI and CBMS symposia would not have been possible without the gracious support of Matt Lane, Cheryl McFadden, Angie Young, Dr. Mike Brent, Candice Adams and Yvonne Kline. I also give thanks to other members of the Department of Chemistry; Andrea Carroll, Christine Zay and Mandy Swope and especially Judith Currano. iv As a protein crystallographer, I had the unique opportunity to take numerous synchrotron data collection trips, where I not only collected data, but also learned extensively about new techniques and software. I thank the Cornell High Energy Synchrotron Source, the National Light Source at Brookhaven National Laboratories, and the Advanced Photon Source at Argonne National Laboratories, especially the beam line scientists at NE-CAT for access to X-ray facilities. This work was supported by NIH grants GM49758, GM56838 and a NIH CBI Training Grant. I would like to thank the members of my graduate class, with whom I have had a lot of fun these past 5 years. Firstly I would like to thank Dr. Dan Himmelberger, my closest friend, for his love, support and patience. Thanks to Sara (Nichols) Hayik and Ian Farrell, whom together with Dan, have been my lunch group for the past 4 years – these are some of my best memories. My thanks go to Deidre Sandrock, Diana (Cabral) Challen, Maria (Gilleece) Bednar, Jenny (Muth) Chambers, Alice Chong, Andre Isaacs, Belgin Canturk and Ken Lassen – what a fantastic group of students to start with at Penn. I would also like to thank Ariane Perez-Gavilian, Liz Roesser, Brandon Kelley, Danielle Reifsnyder, Rob Hickey, Ben Dyme, Dave Perez, and especially Emily Berkeley for their friendship. I would also like to thank my girlfriends from Canada who have been nothing but supportive, although they never really understood what I was up to; Dr. Sabrina Akhtar, Dr. Kristina Powles, Dr. Hinal Sheth and Dr. Monica Hau. v Finally, I would like to thank my family for their incredible support. First, to my New Jersey family-away-from-home, a special thanks to Aunt Judy and Uncle Mike Marinelli and Aunt Char and Uncle Phil McMorris who always treated me as one of their own. To my grandparents, Helen Freund (Gram), and
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