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dc.contributor.authorhuang, hua
dc.date.accessioned2012-02-01T18:14:52Z
dc.date.available2012-02-01T18:14:52Z
dc.date.issued2012-02-01
dc.date.submittedDecember 2011
dc.identifier.urihttp://hdl.handle.net/1928/17480
dc.description.abstractThe 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 -phosphoglucomutase(-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.en_US
dc.language.isoenen_US
dc.subjectHAD Superfamilyen_US
dc.subjectPyrophosphataseen_US
dc.subjectKDO8PPen_US
dc.subjectFkbHen_US
dc.subject.lcshHaloacid dehalogenase--Structure-activity relationships.
dc.titleFunction assignment within the haloacid dehalogenase superfamilyen_US
dc.typeDissertationen_US
dc.description.degreeChemistryen_US
dc.description.levelDoctoralen_US
dc.description.departmentUniversity of New Mexico. Dept. of Chemistryen_US
dc.description.advisorDunaway-Mariano, Debra
dc.description.committee-memberKirk, Martin
dc.description.committee-memberMariano, Patrick
dc.description.committee-memberAllen, Karen


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