University of New Mexico UNM Digital Repository Chemistry ETDs Electronic Theses and Dissertations 2-1-2012 Function assignment within the haloacid dehalogenase superfamily hua huang Follow this and additional works at: https://digitalrepository.unm.edu/chem_etds Recommended Citation huang, hua. "Function assignment within the haloacid dehalogenase superfamily." (2012). https://digitalrepository.unm.edu/ chem_etds/22 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Chemistry ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. FUNCTION ASSIGNMENT WITHIN THE HALOACID DEHALOGENASE SUPERFAMILY BY HUA HUANG DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Chemistry The University of New Mexico Albuquerque, New Mexico December, 2011 ACKNOWLEDGEMENTS Foremost, I would like to express my greatest and sincerest gratitude to my research advisor- Professor Debra Dunaway-Mariano. It is she who opened the door for my exploration of biochemistry world, and during this journey, she always expressed her insight in knowledge and patience in teaching. With the help of her sustained encouragement and guidance, I successfully overcame the difficulties one by one. I believe the benefits I gained will live for my entire life. The second person I appreciate much is Professor Patrick S. Mariano. Dr. Mariano is a nice and erudite professor. He granted me a bunch of useful advices in the inhibitor and substrate synthesis. I am also very grateful for having Professor Martin L. Kirk, Professor Karen N Allen in my doctoral committee and wish to thank them for their precious time in reading and evaluating my dissertation. I also would like to extend my thanks for the collaborators-Dr. Bill Lu from Boston University and Dr. Rafael Toro from Albert Einstein Lab who helped us to solve the structures of the HAD enzymes. The last but not the least is the current and ex-members in Professor Debra Dunaway-Mariano and Patrick Marino’s research group. They are my colleagues and my friends. My appreciation covered Dr. Liangbing Wang, Dr. Zhimin Li, Dr. Jian Cao, Dr. Chunchun zhang, Dr. Jiwen zou, Dr. Hong Zhao, Dr. Danqi Chen, Mr. Min Wang, Ms. Li zheng, Mr. Jonh lathan, Ms. Sarah Toews …….. .Among them, I want to point out my iii special thanks to Dr. Liangbing Wang. He helped me start in my research work and shared his own research experience with me. I benefit a lot from his diligence and experience. My conserved gratitude is left to my wife, Xinshuai Zhang. She always strongly supports me during my Ph.D period. Without her, the life here will become gloomy and the research work should be even tough. Finally, my special thanks go to my family: my parents and sister. Without their support, none of these would be possible. iv FUNCTION ASSIGNMENT WITHIN THE HALOACID DEHALOGENASE SUPERFAMILY BY HUA HUANG ABSTRACT OF DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Chemistry The University of New Mexico Albuquerque, New Mexico December, 2011 FUNCTION ASSIGNMENT WITHIN THE HALOACID DEHALOGENASE SUPERFAMILY HUA HUANG B.S., Chemistry, Lanzhou University, 2002 Ph.D., Chemistry, University of New Mexico, 2011 ABSTRACT The HaloacidDehalogenase Enzyme Superfamily (HADSF) is a ubiquitous family of enzymes.Presently, more than 45, 000 deposited gene sequences encode proteins of the HADSF, and only a fraction of these have defined structure and/or function. The work described in this thesis focuses on function determination in several members of the HADSF. An integrated bioinformatic-protein structure-enzyme mechanism approach was used to differentiate and track D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GHMB) and histidinol phosphatephosphatase (HisB)orthologues;2-keto-3- deoxynononic acid 9-phosphatephosphatase (KDN9PP) and 2-keto-3-deoxy-D-manno- octulosonic acid 8-phosphatephosphatase (KDO8PP)orthologues; inorganic pyrophosphatase and β-phosphoglucomutase (β-PGM) orthologues. In addition, a structure-function/bioinformatic analysis was carried-out on thebifunctional 1,3- diposphoglycerate acyltransferase/Cys-S-glyceryl-3-phosphate phosphatase (FKBH). vi Each study began with the examination of the genome context of the encoding gene of the target HADSF member. Based on this analysis possible catalytic functions were posited. In vitro activity assays were then applied to test possible substrates. Having identified a potential physiological substrate the X-ray structure of the enzyme-substrate (or substrate analog) complex was determined. From this structure the substrate recognition residues were identified. These residues were replaced by site directed mutagenesis and the impact on substrate binding and catalysis was determined by measuring the steady-state kinetic constants kcatand kcat/Km for each of the mutant enzymes. Residues shown to be important were used as sequence markers to identify among the sequence homologues identified in BLAST searches the most confidently defined orthologues. The final step used in the function annotation procedure was to examine the genome context of the encoding gene of each putative orthologue. These data were then used to formulate the proposal for in vivo function. vii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………….XII LIST OF TABLES…………………………………………………..………………XXI LIST OF ABBREVIATIONS……………………………………………….……XXIV CHAPTER ONE: 1HALOACID DEHALOGENASE ENZYME SUPERFAMILY FUNCTION DISCOVERY ...............................................................................................1 1.1 Introduction to the Haloacid Dehalogenase Enzyme Superfamily ..........................1 1.2 The Structural Determinants of HADSF Phosphatases Substrate Recognition .....11 1.3 HADSF Phosphatase Function Determination .......................................................16 1.4 Reference: ...............................................................................................................18 CHAPTER TWO: DIVERGENCE OF STRUCTURE AND FUNCTION IN THE HALOACID DEHALOGENASE (HAD) ENZYME SUPERFAMILY: BACTERIODES THETAIOTAMICRON BT2127 IS AN INORGANIC PYROPHOSPHATASE ..................................................................................................20 2.1 Introduction ............................................................................................................20 2.2 Materials and Methods ...........................................................................................23 2.2.1 General Method .............................................................................................23 2.2.2 Preparation of Recombinant Wild-type and Mutant BT2127 .......................23 2.2.3 BT2127 Molecular Weight Determination ....................................................25 2.2.4 Kinetic Assay for Beta-Phosphoglucomutase Activity .................................26 viii 2.2.5 Steady-State Kinetic Constant Determinations .............................................26 2.2.6 pH Rate Profile Determination ......................................................................27 2.2.7 BT2127 Crystallization and Structure Determination ...................................27 2.2.8 Bioinformatic Analysis ..................................................................................29 2.3 Results and Discussion ...........................................................................................30 2.3.1 BT2127 Substrate Specificity Profile ............................................................30 2.3.2 BT2127 Structure Determination ..................................................................35 2.3.3 Site Directed Mutagenesis of BT2127 Active Site and Domain-Domain Interface Residues ..................................................................................................52 2.3.4 BT2127 Biological Range and In Vivo Function ..........................................63 2.3.5 Structural Determinants in the Divergence of Function in BT2127 and ß- PGM .......................................................................................................................68 2.3.6 Evolution of Pyrophosphatases Within the HADSF and Other Superfamilies ..............................................................................................................................71 2.3.7 Conclusion .....................................................................................................77 2.4 References ..............................................................................................................78 CHAPTER THREE: STRUCTURE, CATALYSIS AND FUNCTION OF FKBH ..83 3.1 Introduction ............................................................................................................83 3.2 Materials and Methods ...........................................................................................88 3.2.1 General Methods ...........................................................................................88 3.2.2 Preparation of Wild-Type Recombinant BF0824 (ExPasY #Q64Y51) ........89 3.2.3 Preparation of Wild-Type Recombinant BF1531 (ExPasY # Q64W45) ......92 3.2.4 Preparation of BF0824 Site Directed Mutants and Truncation Mutants .......93 ix 3.2.5 Preparation of the Apo and Holo Acyl Carrier Proteins BF0825 (ExPasY #Q64Y50) and BF0819 (ExPasY #Q64Y56) .........................................................95