Dissertation/Thesis Abstract

Function assignment within the haloacid dehalogenase superfamily
by Huang, Hua, Ph.D., The University of New Mexico, 2011, 212; 3504257
Abstract (Summary)

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). 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 kcat and kcat/K m 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.

Indexing (document details)
Advisor: Dunaway-Mariano, Debra
Commitee: Allen, Karen N., Kirk, Martin L., Mariano, Patrick S.
School: The University of New Mexico
Department: Chemistry and Chemical Biology
School Location: United States -- New Mexico
Source: DAI-B 73/08(E), Dissertation Abstracts International
Subjects: Chemistry, Biochemistry
Keywords: Fkbh, Had superfamily, Haloacid dehalogenase, Kdn9pp, Kdo8pp, Phosphatase, Pyrophosphatase
Publication Number: 3504257
ISBN: 978-1-267-28308-5
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