Chemistry and Chemical Biology ETDs

hua huang

Publication Date

2-1-2012

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 the bifunctional 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 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.

Language

English

Keywords

HAD Superfamily, Pyrophosphatase, KDO8PP, FkbH

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Kirk, Martin

Second Committee Member

Mariano, Patrick

Third Committee Member

Allen, Karen

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