STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF ENZYMES IN COG3964 OF THE AMIDOHYDROLASE SUPERFAMILY: FROM SEQUENCE TO STRUCTURE TO FUNCTION A Dissertation by ARGENTINA ORNELAS Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Frank M. Raushel Committee Members, Wenshe Liu Donald W. Pettigrew Coran M.H. Watanabe Head of Department, David H. Russell December 2012 Major Subject: Chemistry Copyright 2012 Argentina Ornelas ABSTRACT The Amidohydrolase Superfamily (AHS) of enzymes is one of the most structurally and functionally studied groups of biological catalysts, exquisitely designed to carry out an extensive number of reactions defined by a similar reaction mechanism. There are approximately 11,000 genes coding for AHS proteins from about 2,100 sequenced organisms. Sequence information for these genes has been catalogued in databases, the most instrumental being the National Center for Biotechnology Information (NCBI). Despite the accessible information organized in genomic databases, there is still an extensive problem of reliability in the functional annotation of gene products assigned to the AHS. Proteins in COG3964 of the AHS have been functionally identified as dihydroorotases and adenine deaminases. Eight proteins within three group families of COG3964 have been purified and fail to demonstrate the functionally annotated activity. A library of compounds developed by functional-group modifications was compiled and tested with these enzymes. A group of enzymes within COG3964 demonstrates the ability to hydrolyze stereospecific acetylated -hydroxyl carboxylates. Substrate profiles were constructed for enzymes belonging to group 6 of COG3964. Atu3266, Oant2987 and RHE_PE00295 hydrolyze the R-isomers of a library of -acetyl carboxylates of which acetyl-R-mandelate is the best substrate with catalytic efficiencies of 105 M-1s-1. This compound was identified after a series of modifications from a low-activity compound (V/K = 4 M-1s-1). Methylphosphonate analogs of acetyl-R-mandelate and N- ii acetyl-D-phenyl glycine are inhibitors of enzymes in group 6. The structure of Atu3266 was used in docking experiments to assess the selectivity of R- enantiomers over their S- counterparts. An additional group of orthologues share less than 40% sequence similarity to enzymes from group 6. EF0837, STM4445 and BCE_5003 from group 2 show significantly lower rates for the hydrolysis of -acetyl carboxylates, including 3 -1 -1 acetyl-R-mandelate, hydrolyzed at values of kcat/Km = 10 M s . This is also the only active compound for EF0837. Xaut_0650 and blr3349 from group 7 of COG3964 demonstrate less than 30% identity to enzymes in groups 2 and 6. These enzymes fail to hydrolyze any compound from an extended library of compounds. An annotated selenocysteine synthase gene (SelA) from COG1921 has been identified as a gene neighbor to almost every amidohydrolase from COG3964. Atu3263, Oant2990 and EF0838 are pyridoxal-5’-phosphate dependent enzymes that were purified and assayed with D- and L- amino acids. Initial thermal-shift fluorescence assays determined that in the presence of D-cysteine, the proteins were denatured at lower temperatures. iii DEDICATION A mi familia, por su apoyo y amor – Los quiero mucho. To my family, for their love and support. iv ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Frank M. Raushel, for his guidance and support throughout the course of this research, and my committee members: Dr. Donald Pettigrew, Dr. Wenshe Liu, and Dr. Coran Watanabe. I would also like to extend my appreciation to Dr. Tamari Narindoshvili, Dr. Magdalena Korczynska, and to past and present members of the Raushel Lab, the best ‘superfamily’ that I could ask for in grad school. Thanks to my family, a big crazy bunch of people who supported me and had confidence in me, and to Michael Dearing, for his support and understanding through this phase of my career. Finally, thanks to Arthur Kornberg’s For the Love of Enzymes, a book that provided me with inspiration during the tough times of graduate school. v NOMENCLATURE NCBI National Center for Biotechnology Information SCOP Structural Classification of Proteins PDB Protein Data Bank GenBank Gene Bank database UniProtKB Universal Protein resource Knowledge Base TIM TrIose phosphate isoMerase PSI-BLAST Position-Specific Iterative Basic Local Alignment Search Tool AHS AmidoHydrolase Superfamily M, M Metal at -position and -position of active site COG Cluster of Orthologous Groups EFI Enzyme Function Initiative HEI High Energy Intermediate KEGG Kyoto Encyclopedia for Genes and Genomes ATCC American Type Culture Collection NYSGXRC New York Structural GenomiX Research Consortium SDS-PAGE Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis MUSCLE MUltiple Sequence Comparison by Log-Expectation SelA Selenocysteine synthase PLP Pyridoxal-5’-Phosphate vi TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. ii DEDICATION .......................................................................................................... iv ACKNOWLEDGEMENTS ...................................................................................... v NOMENCLATURE .................................................................................................. vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... ix LIST OF TABLES .................................................................................................... xii CHAPTER I INTRODUCTION .......................................................................... 1 CHAPTER II FUNCTIONAL ANNOTATION AND THREE-DIMENSIONAL STRUCTURE OF INCORRECTLY ANNOTATED DIHYDROOROTASES FROM COG3964 IN THE AMIDOHYDROLASE SUPERFAMILY ..................... 40 Materials and Methods ........................................................................................ 44 Results ….. ........................................................................................................ 59 Discussions… ...................................................................................................... 79 CHAPTER III STRUCTURAL STUDIES, SUBSTRATE DIVERSITY AND FUNCTIONAL ANNOTATION OF ORTHOLOGUES IN COG3964 ENZYMES: INSIGHTS FROM EF0837, BCE_5003 AND STM4445 .................. 97 Materials and Methods ........................................................................................ 102 Results……… ..................................................................................................... 111 Discussions.... ...................................................................................................... 134 CHAPTER IV FUNCTIONAL DIVERSITY IN COG 3964: SEARCHING AND ASSESSING THE FUNCTIONAL ROLES OF OTHER AMIDOHYDROLASES .......................................................................................... 140 Materials and Methods ........................................................................................ 147 Results and Discussions….. ................................................................................ 149 vii Page CHAPTER V INSIGHTS INTO OPERON PROTEINS FOR FUNCTIONAL ANNOTATION OF ENZYMES IN COG3964: ASSESSING A FUNCTIONAL RELATIONSHIP BETWEEN COG3964 AND COG1921 ...................................... 167 Materials and Methods ........................................................................................ 174 Results… ............................................................................................................. 181 Discussions ......................................................................................................... 186 CHAPTER VI SUMMARY AND CONCLUSIONS ............................................. 196 REFERENCES .......................................................................................................... 208 viii LIST OF FIGURES FIGURE Page 1.1 Graph relating sequence to function similarities according to Gerstein .... 4 1.2 Structure of (/)8-barrel of triosephosphate isomerase ............................ 10 1.3 Active site subtypes in the AHS of enzymes ............................................. 17 1.4 Functional misannotation in the AHS ........................................................ 20 1.5 Sequence similarity network of twenty-four COGs in the AHS ................ 26 1.6 Sequence similarity network of COG3964 ................................................ 28 1.7 Phylogenetic profiles of organisms with COG3964 enzymes ................... 33 1.8 Sequence similarity network of COG1921 ................................................ 35 2.1 Sequence similarity network of COG3964 with group 6 enzymes ............ 43 2.2 Ribbon representation of the hexameric structure of Atu3266 .................. 62 2.3 Ribbon representation of the monomeric structure of Atu3266 ................. 63 2.4 Active site of Atu3266 ............................................................................... 64 2.5 Comparison of rates in hydrolysis of selected compounds ....................... 69 2.6 Inhibition curves for activity of Atu3266 and Oant2987 ........................... 70 2.7 Docking results and models of N-acetyl-D-/L-amino acids .......................