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

AgaR, 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_glpK, 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_glpK. 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_m (SSO₃²⁻) and K_m (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_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Anaerobic Microbiology

Department dc:contributor.department

Biochemistry and Anaerobic Microbiology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1999

Author dc:creator

• Ray, William Keith

Chair dc:contributor.committeechair

• Larson, Timothy J.

Committee members dc:contributor.committeemember

• Chen, Jiann-Shin
• Grabau, Elizabeth A.
• Stevens, Ann M.
• Bevan, David R.

• sulfurtransferase
• iron-sulfur cluster biosynthesis
• repressors
• carbon metabolism
• transcriptional control

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-082099-094958

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/28740