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FUNCTIONAL EXPLORATION and CHARACTERIZATION of the DEAMINASES of COG0402 a Dissertation by DANIEL STEPHEN HITCHCOCK Submitted To

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FUNCTIONAL EXPLORATION and CHARACTERIZATION of the DEAMINASES of COG0402 a Dissertation by DANIEL STEPHEN HITCHCOCK Submitted To FUNCTIONAL EXPLORATION AND CHARACTERIZATION OF THE DEAMINASES OF COG0402 A Dissertation by DANIEL STEPHEN HITCHCOCK Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Frank M. Raushel Committee Members, Tadhg Begley Vishal Gohil Paul Straight Head of Department, Gregory Reinhart May 2014 Major Subject: Biochemistry Copyright 2014 Daniel Stephen Hitchcock ABSTRACT High throughput sequencing technology and availability of this information has changed the way enzyme families can be studied. Sequence information from large public databases such as GenBank and UniProtKB can easily retrieved for the purpose of identifying unique enzymatic activities. The strategy adopted for this study is to identify characterized enzymes and the sequence features which give rise to their substrate specificity. Homologues of these enzymes are retrieved, and any active site variations can be readily identified. Cluster of Orthologous Groups (cog) 0402 is a family of enzymes which comprise a portion of the amidohydrolase superfamily. This group catalyzes a deamination reaction, releasing free ammonia and replacing it with a tautomerized oxygen. Cog0402 is most well known for guanine and cytosine deaminase, however other functions exist. One such function was that of S- adenosylhomocysteine deaminase, which was related to a large group of uncharacterized enzymes. These enzymes were predicted by us to deaminate 5’- modified adenosines. The enzymes were physically characterized these predictions were confirmed and a 5’-deoxyadenosine deaminase was discovered in addition to an 8-oxoadenine deaminase. During this study it was noted that background isoguanine deaminase activity was found at appreciable rates in E. coli. This activity was purified and identified using nanoLC-MS/MS and found to be caused by E. coli cytosine ii deaminase. E. coli cytosine deaminase itself is found in a cluster of uncharacterized enzymes with a single amino acid difference in the active site. Representative enzymes were purified and a 5-methylcytosine deaminase was discovered. This enzyme is capable of rescuing thymine auxotrophs in the presence of 5-methylcytosine, and will confer sensitivity to 5-fluorocytosine. Finally, an enzyme distantly related to cytosine deaminase was purified and found to be a unique pterin deaminase. It was most efficient for oxidized pterin rings and would accept a variety of substituents on the C6 positions. Futhermore, it was thought to catalyze the first step of an undescribed pterin degradation pathway. iii DEDICATION To my Dad, who always inspired me to approach questions rationally and scientifically. To my Mom, who provided me with the unconditional love that only a mother could show. To my Brothers Bill and Tommy who gave me friendship. And to my Neice and Nephew, whose curiosity of the world around them reminds me why I became interested in science. iv ACKNOWLEDGEMENTS This work would not have been possible without the help of my advisor, my committee, my collaborators, my lab mates, and the various faculty members who used their time to help me. Dr. Raushel has provided me with all the tools guidance and encouragement I could use in a research lab. He provided me clear goals on what to do, however always was open to new ideas and gave me the creative freedom to explore projects which interested me the most. Both Dr. Manson and Dr. Dangott provided technical advice which made my projects possible. Dr. Gohil was very generous in using his contacts and assisting me with continuing my career after I finish at Texas A&M. My lab members were always friendly, provided me with advice and were particularly patient whenever my experiments were not cleaned up in a timely manner. Also, a special acknowledgement goes to the Texas A&M Triathlon team. The group always provided me with friendship, a hobby outside of work, and helped me feel like an athlete again at times when when my days were spent sitting in lab. I gained confidence and physical health by racing which carried into my research, and my time in graduate school was enriched by the youthful energetic students on the team. v TABLE OF CONTENTS Page ABSTRACT.............................................................................................................. ii DEDICATION.......................................................................................................... iv ACKNOWLEDGEMENTS......................................................................................... v TABLE OF CONTENTS............................................................................................. vi LIST OF FIGURES.................................................................................................... viii LIST OF TABLES...................................................................................................... xi CHAPTER I INTRODUCTION: COG0402 OF THE AMIDOHYDROLASE SUPERFAMILY, NON-AMIDOHYDROLASE DEAMINASES AND THE AMIDOHYDROLASES OF HUMANS................................................. 1 II STRUCTURE-GUIDED DISCOVERY OF NOVEL ENZYME FUNCTIONS: FUNCTIONAL DIVERSITY IN GROUP 1 OF COG0402........................................................................................ 35 Introduction....................................................................... 35 Methods............................................................................. 39 Results................................................................................ 45 Discussion.......................................................................... 55 III RESCUE OF THE ORPHAN ENZYME ISOGUANINE DEAMINASE...... 66 Introduction....................................................................... 66 Results and Discussion....................................................... 67 IV DISCOVERY OF A 5-METHYLCYTOSINE DEAMINASE...................... 76 Introduction....................................................................... 76 Materials and Methods...................................................... 79 Results................................................................................ 83 vi Page Discussion.......................................................................... 91 Conclusion.......................................................................... 93 V ASSIGNMENT OF PTERIN DEAMINASE ACTIVITY TO AN ENZYME OF UNKNOWN FUNCTION GUIDED BY HOMOLOGY MODELING AND DOCKING............................................................. 95 Introduction....................................................................... 95 Materials and Methods...................................................... 98 Results................................................................................ 108 Discussion.......................................................................... 117 Conclusion.......................................................................... 123 VI ATTEMPTS TO ASCERTAIN THE ENZYMATIC ACTIVITY OF E. coli GENES SSNA AND YAHJ.................................................................. 124 Introduction………………………………………………………………..... 124 Results and Discussion……………………………………………......... 124 VII CONCLUSION.................................................................................. 132 REFERENCES........................................................................................................... 137 APPENDIX A........................................................................................................... 168 APPENDIX B........................................................................................................... 182 APPENDIX C............................................................................................................ 186 vii LIST OF FIGURES FIGURE Page 1.1 The active site of members of cog0402.................................................... 3 1.2 Cog0402 rendered at an E-value of 10-50.................................................. 5 1.3 The active site overlay of cytosine and guanine deaminase..................... 6 1.4 Network diagram of members of cog0295 rendered at a BLAST E-value of 10-30...................................................................................................... 10 1.5 The active site schematics of various members of cog0295..................... 11 1.6 Network diagrams of cog0717.................................................................. 15 1.7 Active site of PDB: 1XS1............................................................................ 17 1.8 Structural comparison of a group 4 protein and a homodimer from group 1...................................................................................................... 19 1.9 A) Active site of 2HVW B) Active site of 1VQ2.......................................... 20 1.10 Allosteric site of 2HVW.............................................................................. 21 1.11 Cog2131.................................................................................................... 22 1.12 Cog0590 at 10-30........................................................................................ 25 1.13 Active site shown in PBD: 2B3J.................................................................. 27 1.14 Active site of 1WKQ with modelled guanine-related inhibitor………………. 27 1.15 Active site of 1P6O...................................................................................
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