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D-AMINOACYLASES AND DIPEPTIDASES WITHIN THE AMIDOHYDROLASE SUPERFAMILY: RELATIONSHIP BETWEEN ENZYME STRUCTURE AND SUBSTRATE SPECIFICITY A Dissertation by JENNIFER ANN CUMMINGS 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 December 2010 Major Subject: Chemistry D-AMINOACYLASES AND DIPEPTIDASES WITHIN THE AMIDOHYDROLASE SUPERFAMILY: RELATIONSHIP BETWEEN ENZYME STRUCTURE AND SUBSTRATE SPECIFICITY A Dissertation by JENNIFER ANN CUMMINGS 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 Raushel Committee Members, Paul Lindahl David Barondeau Gregory Reinhart Head of Department, David Russell December 2010 Major Subject: Chemistry iii ABSTRACT D-Aminoacylases and Dipeptidases within the Amidohydrolase Superfamily: Relationship Between Enzyme Structure and Substrate Specificity. (December 2010) Jennifer Ann Cummings, B.S., Southern Oregon University; M.S., Texas A&M University Chair of Advisory Committee: Dr. Frank Raushel Approximately one third of the genes for the completely sequenced bacterial genomes have an unknown, uncertain, or incorrect functional annotation. Approximately 11,000 putative proteins identified from the fully-sequenced microbial genomes are members of the catalytically diverse Amidohydrolase Superfamily. Members of the Amidohydrolase Superfamily separate into 24 Clusters of Orthologous Groups (cogs). Cog3653 includes proteins annotated as N-acyl-D-amino acid deacetylases (DAAs), and proteins within cog2355 are homologues to the human renal dipeptidase. The substrate profiles of three DAAs (Bb3285, Gox1177 and Sco4986) and six microbial dipeptidase (Sco3058, Gox2272, Cc2746, LmoDP, Rsp0802 and Bh2271) were examined with N-acyl-L-, N-acyl-D-, L-Xaa-L-Xaa, L-Xaa-D-Xaa and D-Xaa-L-Xaa substrate libraries. The rates of hydrolysis of the library components were determined by separating the amino acids by HPLC and quantitating the products. Gox1177 and Sco4986 hydrolyzed several N-acyl-D-amino acids, especially those where the amino acid was a hydrophobic residue. Gox1177 hydrolyzed L-Xaa-D- iv Xaa and N-acetyl-D-amino acids with similar catalytic efficiencies (~104 M-1s-1). The best substrates identified for Gox1177 and Sco4986 were N-acetyl-D-Trp and N-acetyl- D-Phe, respectively. Conversely, Bb3285 hydrolyzed N-acyl-D-Glu substrates (kcat/Km ≈ 5 x 106 M-1s-1) and, to a lesser extent, L-Xaa-D-Glu dipeptides. The structure of a DAA from A. faecalis did not help explain the substrate specificity of Bb3285. N- methylphosphonate derivatives of D-amino acids were inhibitors of the DAAs examined. The structure of Bb3285 was solved in complex with the N-methylphosphonate derivative of D-Glu or acetate/formate. The specificity of Bb3285 was due to an arginine located on a loop which varied in conformation from the A. faecalis enzyme. In a similar manner, six microbial renal dipeptidase-like proteins were screened with 55 dipeptide libraries. These enzymes hydrolyzed many dipeptides but favored L-D dipeptides. Respectable substrates were identified for proteins Bh2271 (L-Leu-D-Ala, 4 -1 -1 5 -1 -1 kcat/Km = 7.4 x 10 M s ), Sco3058 (L-Arg-D-Asp, kcat/Km = 7.6 x 10 M s ), Gox2272 5 -1 -1 5 - (L-Asn-D-Glu, kcat/Km = 4.7 x 10 M s ), Cc2746 (L-Met-D-Leu, kcat/Km = 4.6 x 10 M 1 -1 5 -1 -1 s ), LmoDP (L-Leu-D-Ala, kcat/Km = 1.1 x 10 M s ), Rsp0802 (L-Met-D-Leu, kcat/Km = 1.1 x 105 M-1s-1). Phosphinate mimics of dipeptides were inhibitors of the dipeptidases. The structures of Sco3058, LmoDP and Rsp0802 were solved in complex with the pseudodipeptide mimics of L-Ala-D-Asp, L-Leu-D-Ala and L-Ala-D-Ala, respectively. The structures were used to assist in the identification of the structural determinants of substrate specificity. v ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Frank Raushel, for his guidance, patience and support, and my committee members, Dr. Lindahl, Dr. Barondeau, and Dr. Reinhart. Thanks to my former lab mates, Tamiko Porter and Sarah Aubert, and comrades, Eman Ghanem and LaKenya Williams for showing me the ropes during the early days. I would also like to express my appreciation for Jinny Johnson from the Protein Chemistry Laboratory at Texas A&M University for providing the amino acid analysis service and her gracious acceptance of my habitual perfectionism. Finally, I would like to thank my family and friends for their encouragement and love. vi NOMENCLATURE a.a. One of the twenty standard amino acids except cysteine AHS AmidoHydrolase Superfamily ATP Adenosine TriPhosphate BLAST Basic Local Alignment Search Tool 1-8 Numbers 1 through 8 of the -strands within the (/)8-barrel Hepes 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid HPLC High-Performance Liquid Chromatography ICP-MS Inductively Coupled Plasma Mass Spectrometry KEGG Kyoto Encyclopedia of Genes and Genomes M Alpha Metal M Beta Metal NAD+ -Nicotinamide Adenine Dinucleotide NADH -Nicotinamide Adenine Dinucleotide reduced form NCBI National Center for Biotechnology Information NYSGXRC New York Structural Genomics Research Center OD Optical Density OPA o-Phthalaldehyde SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Tris Tris(hydroxymethyl)aminomethane Xaa One of the twenty standard amino acids except cysteine vii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii ACKNOWLEDGEMENTS ...................................................................................... v NOMENCLATURE .................................................................................................. vi TABLE OF CONTENTS .......................................................................................... vii LIST OF FIGURES ................................................................................................... ix LIST OF TABLES .................................................................................................... xiii CHAPTER I INTRODUCTION AND LITERATURE REVIEW ............................ 1 II DEACYLATION OF D-AMINO ACIDS BY MEMBERS OF THE AMIDOHYDROLASE SUPERFAMILY ........................................... 29 Introduction .................................................................................... 29 Materials and Methods ................................................................... 34 Results ............................................................................................ 46 Discussion ...................................................................................... 68 III SUBSTRATE PROFILE AND STRUCTURE OF Sco3058: THE CLOSEST MICROBIAL HOMOLOGUE TO THE HUMAN RENAL DIPEPTIDASE ...................................................................... 80 Introduction .................................................................................... 80 Materials and Methods ................................................................... 83 Results ............................................................................................ 89 Discussion ...................................................................................... 101 IV SUBSTRATE PROFILES AND NATIVE STRUCTURES OF TWO MICROBIAL RENAL DIPEPTIDASE-LIKE PROTEINS. ..... 111 Introduction .................................................................................... 111 Materials and Methods ................................................................... 115 viii CHAPTER Page Results ............................................................................................ 123 Discussion ...................................................................................... 136 V THREE RENAL DIPEPTIDASE-LIKE PROTEINS WITH AN ACTIVE SITE TRYPTOPHAN. ......................................................... 144 Introduction .................................................................................... 144 Materials and Methods ................................................................... 147 Results ............................................................................................ 153 Discussion ...................................................................................... 177 VI CONCLUSIONS .................................................................................. 186 Summary ........................................................................................ 186 D-Aminoacylases in cog3653 ......................................................... 187 Microbial Homologues to the Human Renal Dipeptidase.............. 189 Genome Context ............................................................................. 192 REFERENCES .......................................................................................................... 194 VITA ......................................................................................................................... 204 ix LIST OF FIGURES FIGURE Page 1.1 Genome context of amidohydrolase superfamily members with recently elucidated functions ................................................................... 6 1.2 Stick representations of the ten metal class centers of the amidohydrolase superfamily ................................................................... 18 1.3 Proposed mechanism of the E. coli dihydroorotase and uronate isomerase ................................................................................................
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