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SUN-DISSERTATION-2019.Pdf ENGINEERING IODOTYROSINE DEIODINASE TOWARDS BIOREMEDIATION OF HALOPHENOLS AND CHARACTERIZATION OF A UNIQUE THERMOPHILIC IODOTYROSINE DEIODINASE FROM Thermotoga neapolitana by Zuodong Sun A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland October 2019 © 2019 Zuodong Sun All rights reserved Abstract Iodotyrosine deiodinase (IYD) promotes reductive dehalogenation of chloro-, bromo-, and iodotyrosines (Cl-Tyr, Br-Tyr, and I-Tyr, respectively). This activity decreases dramatically for phenolic substrates lacking the zwitterion due to their inability to close an active site lid. Enhancing this activity will provide an attractive approach to bioremediation of halophenols. A combination of computational design with Rosetta, library construction and screening was employed to promote IYD’s activity towards 2-iodophenol (2IP, a model for halophenol). The lid sequence of IYD from Homo sapiens (HsIYD) was redesigned by a fixed backbone approach to stabilize lid closure in the presence of 2IP. This approach successfully yielded a variant UD08 that moderately improved 2IP deiodination by 4.5-fold compared to HsIYD. UD08 expressed a disorder-to-order transition of the lid induced by 2IP as predicted by Rosetta and verified via limited proteolysis. This resembles the induced lid closure by I-Tyr in HsIYD. IYD from Haliscomenobacter hydrossis (HhIYD), as an easy-to-work with alternative to HsIYD, was subsequently redesigned via a fixed backbone approach and a loop remodeling approach to further improve IYD-2IP interactions. Rosetta once again demonstrated stabilization of the targeted enzyme•2IP complex with loop remodeling although an active 2IP deiodinase was not generated from these approaches. IYD from a thermophilic bacteria Thermotoga neapolitana (TnIYD) was characterized for its potential to facilitate engineering and provide a crystal structure of the fully reduced IYD. TnIYD is the smallest iodotyrosine deiodinase characterized to date and therefore represents a minimal structural requirement for reductive dehalogenation. TnIYD exhibits many unique properties different from those of mesophilic IYDs such as the formation of FMN semiquinone during purification and its tight binding of tyrosine (Tyr). The oxidized FMN in crystals of I-Tyr, Tyr, and fluorotyrosine (F-Tyr) bound TnIYD was readily reduced to the FMN semiquinone, but not to the hydroquinone form upon dithionite treatment. High resolution crystal structures of I-Tyr, F-Tyr, and Tyr bound TnIYD with oxidized and semiquinone FMN suggested that no major ii conformational changes from the oxidized structure were needed to support the FMN semiquinone and other radical intermediates generated during catalysis. An additional binding site for I-Tyr was identified on the surface of TnIYD and this might explain the substrate inhibition of IYDs observed under steady-state conditions. The reduction of TnIYD and HsIYD by dithionite was inhibited by both I-Tyr and Cl-Tyr and may cause the reduction of IYD to be rate-determining during steady- state catalysis. Primary Reader and Advisor: Prof. Steven E. Rokita (Department of Chemistry) Secondary Reader: Prof. Craig A. Townsend (Department of Chemistry) Prof. Jeffrey J. Gray (Department of Chemical & Biomolecular Engineering) iii Acknowledgement The work presented in this dissertation was only made possible from countless help and support that I have received over the years. I would first like to thank my advisor Prof. Steve Rokita for making me the scientist who I am today by providing indispensable advising from problem solving techniques to English writing skills. He allowed me to explore my own research interest and treated me like a peer rather than a student when it came to experiment design and result analysis. This level of independence that I was able to acquire is of great value towards my long term goal of becoming an independent investigator. He has been an amazing advisor for his civility, his patience, his understanding, and his unconditional support to every one of his students. He will always be a role model for me and my future career. I would like to thank my committee members Prof. Craig Townsend and Prof. Jeffrey Gray for reading and commenting on my thesis. In addition, I would like to thank them for their help and their recommendation letters for my postdoc application. Many thanks to Prof. Stephen Fried and Prof. Marc Ostermeier for their help with postdoc application as well. In addition, I would like to thank Prof. John Toscano for being in my GBO committee and Prof. Louise Pasternack for all great advises when I first started in the program. Also I really appreciate all the chemistry staff members for their support on research and administrative affairs. Many of the techniques presented in this dissertation are out of the expertise of our lab and would not be possible without our wonderful collaborators in and outside The Johns Hopkins University. Prof. Jeffrey Gray’s insightful course on Rosetta simulation allowed me to access the power of Rosetta as a biochemist without any experience in computational biology. In addition, his lab carried out sophisticated design simulations that are beyond my capabilities. Prof. Jennifer Kavran has offered us tremendous help with her hard work for our protein crystallography, which inspired my great interest in the area. I would also like to acknowledge our new collaborations with Prof. Squire Booker (Pennsylvania State University) on anaerobic crystallography and Prof. iv Giovanni Gadda (Georgia State University) on rapid enzyme kinetics. Through these collaborations I have greatly expanded my expertise. I would like to thank many former Rokita lab members as my mentors. Special thanks to Dr. Shalini Saha and Dr. Nattha Ingavat who helped me the most during my early and later time in the lab. Their skilled and patient demonstrations on lab techniques and always friendly advising are of great value to me. I would also like to thank Dr. Mark Hutchinson, Dr. Jimin Hu, Dr. Arnab Mukherjee, Dr. Blessing Deeyaa, Dr. Abhishek Phatarphekar, and Dr. Petrina Boucher for all the help they offered to me. Many thanks to the current members of the Rokita lab: Danielle Bautista, Shane Byrne, Harry Greenberg, Anton Kozyryev, Daniel Lemen, Ravina Moirangthem, Qi Su, and Bing Xu for their help and support and for the great time we spent together over the years. It is also a great pleasure to work with many talented undergrads and rotation graduate students: Morgan Nance (PMB), Shaun Spisak (CBI), Azam Hussain, and Gregory Berumen. I would like to thank them all for allowing me to have great experiences as a mentor myself. I would like to thank Prof. Hsin Kuo (British Colombia Institution of Technology) for being a wonderful mentor and friend to me when he was a postdoc in our department. His friendship helped me to pass a really difficult time when I faced a major decision for my career. His kind and easygoing charisma is admired by virtually everyone who interacted with him. Many thanks to my friends Dr. Liwei Zheng and Dr. Rongfeng Li as well for their countless help and fun time. I would like to thank the late Prof. Justine Roth for her advising of my research during the first two years of my graduate school. She was a passionate and skilled scientist of shrewd minds. Her board knowledge on many different disciplines of science is the most impressive that I have ever seen. Her great contribution to the scientific community will always be remembered. Last but not the least, I would like to thank my parents for their unconditional love and support. It took great understanding and emotional preparation to send their only child to a foreign country for study. Their love, understanding, and support are indispensable for me to complete my v Ph. D. and explore my dream career path. Many thanks to my other beloved family members: my late grandfather, my grandmother, my aunt and uncle, and my cousins for years of priceless joy and support. vi Dedication To my father Sun, Hongyuan and my mother Liu, Guiping, for their unconditional love and countless support vii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgement ......................................................................................................................... iv Dedication ..................................................................................................................................... vii Table of Contents ........................................................................................................................ viii List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Abbreviations .................................................................................................................. xvi Chapter 1 Introduction.................................................................................................................. 1 1.1 Halophenols: pervasive, persist, and toxic: ....................................................................... 1 1.2 Bioremediation of halophenols: a look at Nature’s toolbox:
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