Characterization of AgaR and YihW, Members of the DeoR Family of Transcriptional Regulators, and GlpE, a Rhodanese Belonging to the GlpR Regulon, Also a Member of the DeoR Family

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dc.contributor.authorRay, William Keithen
dc.contributor.committeechairLarson, Timothy J.en
dc.contributor.committeememberChen, Jiann-Shinen
dc.contributor.committeememberGrabau, Elizabeth A.en
dc.contributor.committeememberStevens, Ann M.en
dc.contributor.committeememberBevan, David R.en
dc.contributor.departmentBiochemistry and Anaerobic Microbiologyen
dc.date.accessioned2014-03-14T20:15:29Zen
dc.date.adate1999-08-24en
dc.date.available2014-03-14T20:15:29Zen
dc.date.issued1999-08-02en
dc.date.rdate2000-08-24en
dc.date.sdate1999-08-20en
dc.description.abstractAgaR, a protein in <i>Escherichia coli</i> thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for <i>agaR, agaZ</i> and <i>agaS</i>, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which <i>agaR</i> had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. <i>In vivo</i> and <i>in vitro</i> analysis of the <i>agaZ</i> promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the <i>aga</i> promoters was less sensitive to catabolite repression in revertants capable of <i>N</i>-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the <i>E. coli</i> genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of <i>sn</i>-glycerol 3-phosphate metabolism in <i>E. coli</i>, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P<sub>glpK</sub>, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P<sub>glpK</sub>. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of <i>sn</i>-glycerol 3-phosphate in <i>E. coli</i> by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K<sub>m</sub> (SSO₃²⁻) and K<sub>m</sub> (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K<sub>m</sub> of 34 mM when thiosulfate was near its K<sub>m</sub>, suggesting thioredoxin may be a physiological substrate.en
dc.description.degreePh. D.en
dc.identifier.otheretd-082099-094958en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-082099-094958/en
dc.identifier.urihttp://hdl.handle.net/10919/28740en
dc.publisherVirginia Techen
dc.relation.haspartEtd.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectsulfurtransferaseen
dc.subjectiron-sulfur cluster biosynthesisen
dc.subjectrepressorsen
dc.subjectcarbon metabolismen
dc.subjecttranscriptional controlen
dc.titleCharacterization of AgaR and YihW, Members of the DeoR Family of Transcriptional Regulators, and GlpE, a Rhodanese Belonging to the GlpR Regulon, Also a Member of the DeoR Familyen
dc.typeDissertationen
thesis.degree.disciplineBiochemistry and Anaerobic Microbiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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