Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid R773.
The ArsC protein encoded by the arsenical resistance operon of plasmid R773 catalyzes the reduction of arsenate to arsenite in Escherichia coli. The reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. The ArsC arsenate reductase has two cysteinyl residues, Cys12 and Cys106. By a combination of random and site-specific mutagenesis, Cys12 was altered to four other amino acid residues. Cells expressing any of those arsC genes were sensitive to arsenate. The ArsCC12S protein was purified and found to be catalytically inactive. Cys106 was altered separately to seryl, glycyl, and valyl residues. Cells expressing arsCC106S, arsCC106G, and arsCC106V genes retained arsenate resistance, and the purified C106S and C106G proteins had reductase activity. Both wild-type ArsC and C106S proteins were inactivated by iodoacetate. In the native enzyme only Cys12 was alkylate by iodoacetate; Cys106 was alkylated only if the enzyme was first denatured. In the presence of the substrate, arsenate, or competitive inhibitors, phosphate or sulfate, the rate of alkylation was reduced. Reductase activity was inhibited by N-ethylmaleimide and could be protected by arsenate. These results suggest Cys12 is an active-site residue essential for catalysis by the arsenate reductase.
Liu
J
J
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Gladysheva
T B
TB
Lee
L
L
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001149:Arsenates / Q000378:metabolism / Q000494:pharmacology; D053501:Arsenite Transporting ATPases; D018053:Arsenites / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D016623:Ion Pumps; D007700:Kinetics; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010084:Oxidation-Reduction; D010641:Phenotype; D011815:R Factors; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D001149:Arsenates; D018053:Arsenites; D016623:Ion Pumps; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D003545:Cysteine