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Download Date 01/10/2021 18:43:00 Wild-type and mutant 2,2-dialkylglycine decarboxylases: The catalytic role of active site glutamine 52 investigated by site-directed mutagenesis and computer analysis Item Type Thesis Authors Woon, See-Tarn Download date 01/10/2021 18:43:00 Link to Item http://hdl.handle.net/11122/9507 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back o f the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Beil & Howell Information Company 300 North Zeeb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. WILD-TYPE AND MUTANT 2,2-DIALKYLGLYCINE DECARBOXYLASES: THE CATALYTIC ROLE OF ACTIVE SITE GLUTAMINE 52 INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND COMPUTER ANALYSIS A THESIS Presented to the Faculty Of the University of Alaska Fairbanks In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY By See-Tam Woon, B. Sc. (Technology) Fairbanks, Alaska August 1998 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9842097 UMI Microform 9842097 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. WILD-TYPE AND MUTANT 2,2-DIALKYLGLYCINE DECARBOXYLASES: THE CATALYTIC ROLE OF ACTIVE SITE GLUTAMINE 52 INVESTIGATED BY SITE-DIRECTED MUTAGENESIS AND COMPUTER ANALYSIS By See-Tam Woon RECOMMENDED: K Advisory Committee Chair < /< Departmental Head ' | APPROVED: c ~Y y u*'y*^~ \ Dean. College of Science, Engineering and Mathematics M = ___________ Detm of Graduate School gr - / / - j x Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT The ability of pyridoxal 5’-phosphate (PLP)-dependent 2,2-dialkylglycine decarboxylase (DGD) to catalyze decarboxylation and transamination of amino acids at a single active site depends on the subsite within the active site that cleaves a-H and a- COCT bonds. As observed in the crystal structure, the strategic position of glutamine 52 at the active site suggests a role in enhancing decarboxylation via formation of a hydrogen bond to the substrate carboxyl group. Supporting evidence for this hypothesis is provided by studies with glutamine 52 active site mutants, computer modeling and protein sequence analyses. Ten mutant DGDs containing alanine, asparagine, aspartate, arginine, glutamate, glycine, histidine, leucine, lysine, and tryptophan at position 52 were produced. All, except the histidine mutant, exhibited decreased rates of decarboxylation compared to wild-type. Histidine and asparagine mutants showed measurable decarboxylation rates. These results and that o f wild-type DGD suggest that hydrogen bonding with the substrate is required for decarboxylation. Mutants incapable of hydrogen bonding to the substrate, such as alanine, leucine and tryptophan mutants, showed negligible decarboxylation reactions. Transamination rates increased for some mutants and decreased for others. These data imply that the DGD subsite is influenced by the presence of glutamine 52. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Furthermore, there is evidence showing that the subsite environment of wild-type DGD, the histidine and the glutamate mutants are different; the three DGD forms exhibited different chromophores at around /.max of 500 nm when treated with 2- methylalanine or L-alanine in the presence of 3% glycerol. These results have important implications for other PLP-dependent enzymes, such as ornithine aminotransferase and y-aminobutyrate aminotransferase. Since protein sequence alignment indicates DGD is homologous to the two aminotransferases, mutations at amino acid position corresponding to glutamine 52 of DGD at the active sites of these aminotransferases could disrupt the functionality of the enzymes. Protein sequence alignment showed that all but one of the PLP-dependent aminotransferases lack residues at position 52 capable of hydrogen bonding with the substrate carboxyl group, further re-affirming the role of glutamine 52 in decarboxylation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS List of Figures............................................................................................................................ viii List of Tables............................................................................................................................... xi List of Appendices ..................................................................................................................... xii List of Abbreviations ................................................................................................................xiii Acknowledgments.....................................................................................................................xiv Chapter 1 Literature review 1 - 31 1.1. Discovery of vitamin B6 ........................................................................................................1 1.2. PLP-catalyzed reactions at a-carbon ................................................................................. 3 1.3. Properties of pyridoxine phosphate ..................................................................................10 1.4. Burkholderia cepacia 2,2-dialkylglycine decarboxylase (DGD) ...........................12-26 1.4.1. Introduction ............................................................................................................... 12 1.4.2. Structural organization of D G D ............................................................................. 15 1.4.3. Substrate and reaction specificity of DGD ............................................................ 20 1.4.4. Normal versus abortive decarboxylation ............................................................... 23 1.5. Significance of conducting DGD research ..................................................................... 26 1.6. Project objectives ................................................................................................................ 27 1.7. Hypothesis testing on how DGD catalyzes decarboxylation ........................................ 28 1.8. Dissertation outline .............................................................................................................30 Chapter 2 Wild-type and mutant 2,2-dialkylglycine decarboxylases: Role of active site Gln52 investigated by site directed mutagenesis 32 - 66 2.1. Abstract................................................................................................................................ 32 2.2. Introduction ..........................................................................................................................34 2.3. Materials and Method ........................................................................................................ 36 2.4. R esults...................................................................................................................................43 V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.5. Discussion ............................................................................................................................55 2.6. Conclusion .......................................................................................................................... 65 Chapter 3 Evolutionary relationships between 2,2-diakyIglycine decarboxylase (DGD) and PLP-dependent enzymes: Investigations into amino acids responsible for DGD decarboxylation ...........................................67 - 90 3.1. Abstract................................................................................................................................67 3.2. Introduction ........................................................................................................................
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