PMID:11032735

Citation	Dyllick-Brenzinger, M, Liu, M, Winstone, TL, Taylor, DE and Turner, RJ (2000) The role of cysteine residues in tellurite resistance mediated by the TehAB determinant. Biochem. Biophys. Res. Commun. 277:394-400
Abstract	TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.
Links	PubMed Online version:10.1006/bbrc.2000.3686
Keywords	Alanine/chemistry; Alkylating Agents/pharmacology; Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Binding Sites; Cysteine/chemistry; Cysteine/physiology; Dithionitrobenzoic Acid/pharmacology; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors/pharmacology; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Ethylmaleimide/pharmacology; Iodoacetamide/pharmacology; Iodoacetates/pharmacology; Ligands; Microbial Sensitivity Tests; Mutagenesis, Site-Directed; Phenotype; Plasmids/metabolism; Protein Binding; Protein Conformation; Protein Denaturation; S-Adenosylmethionine/chemistry; S-Adenosylmethionine/metabolism; Sulfhydryl Reagents/pharmacology; Tellurium/pharmacology; Up-Regulation
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