Purification and characterization of ACR2p, the Saccharomyces cerevisiae arsenate reductase.
In Saccharomyces cerevisiae, expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2 complemented the arsenate-sensitive phenotype of an arsC deletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis of grx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase.
Mukhopadhyay
R
R
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Shi
J
J
Rosen
B P
BP
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053502:Arsenate Reductases; D001149:Arsenates / Q000494:pharmacology; D053501:Arsenite Transporting ATPases; D003001:Cloning, Molecular; D019281:Dimerization; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects; D005816:Genetic Complementation Test; D016623:Ion Pumps; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D001149:Arsenates; D016623:Ion Pumps; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013879:Thioredoxins; C506971:ARR2 protein, S cerevisiae; D053502:Arsenate Reductases; D013880:Thioredoxin-Disulfide Reductase; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases