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Study of Pvca and Pvcb, Two Enzymes Involved in Making Isonitrile

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Study of Pvca and Pvcb, Two Enzymes Involved in Making Isonitrile STUDY OF PVCA AND PVCB, TWO ENZYMES INVOLVED IN MAKING ISONITRILE-CONTAINING NATURAL PRODUCTS IN BACTERIAL PATHOGENS ___________________________________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri-Columbia ______________________________________________________________ In Partial Fulfillment of the Requirements for the Degree Ph.D. in Biochemistry _________________________________________________________________ By Jing Zhu Dr. Peter A. Tipton, Dissertation Advisor DEC 2016 The undersigned, appointed by the dean of the Graduate School, have examined the thesis entitled STUDY OF PVCA AND PVCB, TWO ENZYMES INVOLVED IN MAKING ISONITRILE-CONTAINING NATURAL PRODUCTS IN BACTERIAL PATHOGENS Presented by Jing Zhu A candidate for the degree of doctor of philosophy in Biochemistry, And hereby certify that, in their opinion, it is worthy of acceptance. Dr. Peter A. Tipton Dr. Gerald Hazelbauer Dr. Judy D. Wall Dr. Kent Gates Dr. Timothy Glass ACKNOWLEDGEMENTS The experience living and studying in Columbia, Missouri will become the most precious memory in my whole life and I want to express my gratitude to many people who ever helped me. I would like to thank the Department of Biochemistry at University of Missouri- Columbia for admitting me as a graduate student. Otherwise I would not have this chance to experience different cultures and meet a lot of wonderful people here in the past five years. I would like to extend a special thank you to my thesis advisor Dr. Peter Tipton. I feel so lucky and so appreciative to have a mentor that is very knowledgeable, kind and patient on everyone and everything. His great passion for science and persistence in doing research impressed and inspired me a lot, especially when I felt lost and depressed in the past. He provided me with millions of brilliant ideas about my project but at the same time, taught me how to be an independent thinker and scientist. I have learned a lot from him, both scientifically and personally. I will be thankful for my time spent in Tipton lab forever. I was fortune to work with two very nice ladies Krista Arnett and Dr. Emma Farrell when I joined the lab as a green hand. Krista helped me adapt to all the laboratory techniques and gave me valuable advice on how to use the instruments and how to keep everything organized. Although I only worked with Emma for one year, I was impressed by her kindness, elegance and creativity. I would never ii forget her returning to the lab after she just got home, to open the door for me a couple of times. Thank both of you for guiding and helping me. I want to express my gratitude to the undergraduate students who ever worked in the lab for their companionship. Thank Kennady Gee for his hard work on my project and for teaching me how to be a good mentor. I want to thank my committee members, Dr. Gerald Hazelbauer, Dr. Judy Wall, Dr. Kent Gates and Dr. Timothy Glass for the suggestions and advice they have provided throughout my graduate study. They challenged me to become a better scientist. I would like to thank Yufei Li, Qinyi Wang, Ning Bi, Weiwei Wu, Zhenwei Song, Zhe Li, and Lingyan Jiang for their friendship, support and advice. Without them, life would be boring. Finally, I would like to express my deepest gratitude to my parents, Lianjin Zhu and Yanwei Tang. Their trust, encouragement, support and constant love make me brave, confident and happy. Thank you for being the most important mentors and friends in my life. And thank you for missing me so much like I miss you when I am 20,000 kilometers away from you. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................... ii LIST OF FIGURES .............................................................................................. ix LIST OF TABLES .............................................................................................. xii LIST OF SCHEMES .......................................................................................... xiii LIST OF ABBREVIATIONS .............................................................................. xiv ABSTRACT ..................................................................................................... xviii Chapter 1 General Introduction .......................................................................................... 1 1.1 Isonitriles in Organic Chemistry ................................................................. 2 1.2 Isonitrile-Containing Natural Products ....................................................... 3 1.3 Isonitrile Biosynthesis ................................................................................ 6 1.4 Gene Cluster Related to Isonitrile Biosynthesis ......................................... 8 1.5 Functions of the pvcA/pvcB Gene Clusters and Their Related Metabolites ................................................................................................................. 15 1.6 Structures of the PvcA and PvcB Proteins from P. aeruginosa ................. 18 1.7 Non-Heme Fe(II) and α-Ketoglutarate Dependent Oxygenases ............... 21 1.8 Predicted Activities of the PvcA and PvcB Proteins................................... 23 1.9 Overview of Investigations ........................................................................ 24 iv Chapter 2 Characterization of Three PvcB Homologous Proteins, the Non-Heme Fe2+, α-Ketoglutarate Dependent Oxygenases........................................................ 27 2.1 Published Work ......................................................................................... 27 2.2 Summary of Published Work ..................................................................... 28 2.3 Supplemental Results ............................................................................... 30 2.3.1 Heterologous Expression of PaPvcB, XnPvcB and EaPvcB in E. coli 30 2.3.2 Rapid-Mixing Quench Flow Analysis of the PvcB Reactions ............... 32 2.3.3 Stopped-Flow UV-visible Spectroscopic Analysis of the PvcB Reactions ............................................................................................................ 36 2.3.4 Organic Synthesis and 1H-NMR Characterization of 4-Nitro Phenylalanine Isonitrile and 3-Nitro Tyrosine Isonitrile ........................ 39 2.3.5 PvcB Activity Assays with 4-Nitro Phenylalanine Isonitrile and 3-Nitro Tyrosine Isonitrile .............................................................................. 42 2.4 Conclusion and Discussion ....................................................................... 45 2.5 Material and Methods ................................................................................ 51 2.5.1 SDS-PAGE Analysis of PvcB Expression in E. coli ............................. 51 2.5.2 Rapid Quench Flow Analysis of the PvcB Reactions .......................... 51 2.5.3 Stopped-Flow UV-visible Analysis of the PvcB Reactions .................. 52 2.5.4 Synthesis of 4-Nitro Phenylalanine Isonitrile ....................................... 52 2.5.5 Synthesis of 3-Nitro Tyrosine Isonitrile ................................................ 54 2.5.6 PvcB Activity Assays with 4-Nitro Phenylalanine Isonitrile .................. 55 2.5.7 PvcB Activity Assays with 3-Nitro Tyrosine Isonitrile ........................... 56 2.5.8 HPLC Analyses ................................................................................... 56 v Chapter 3 Characterization of the Reaction Catalyzed by PvcA, an Isonitrile Synthase ........................................................................................................................... 57 3.1 Introduction................................................................................................ 57 3.2 Results ...................................................................................................... 62 3.2.1 Heterologous Expression of PaPvcA and XnPvcA in E. coli ............... 62 3.2.2 Reconstitution of the PvcA Reactions in Vitro ..................................... 64 3.2.3 Reconstitution of the Isocyanovinylphenol Biosynthetic Pathway in E. coli ....................................................................................................... 65 3.2.4 Identification of the Product of the PvcA Reaction .............................. 69 3.2.5 Identification of Tyrosine as One of the Substrates for the PvcA Reaction .............................................................................................. 73 3.2.6 Seeking the Substrate Providing the Carbon for the Isonitrile Group .. 78 3.2.6.1 Tracking of the Isonitrile Carbon in E. coli Expressing AhPvcA- XnPvcB ......................................................................................... 78 3.2.6.2 Searching for the Substrate in a Phosphorylated Metabolite Library and the Major Metabolic Pathways ............................................... 82 3.2.6.3 Looking for the Substrate in the Metabolite Pool of E. coli ............ 83 3.3 Conclusion and Discussion ....................................................................... 94 3.4 Material and Methods .............................................................................. 108 3.4.1 Cloning of PvcA Genes ..................................................................... 108 3.4.2 Heterologous Expression of the PvcA Proteins ................................. 109 3.4.3 Purification of the PvcA Proteins ......................................................
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