Chrysostomou Constantine, Quandt Erik M, Marshall Nicholas M, Stone Everett, Georgiou George
Departments of †Chemical Engineering and ‡Biomedical Engineering, and §Molecular Genetics and Microbiology and Institute for Cell and Molecular Biology, University of Texas, Austin, Texas 78712, United States.
ACS Chem Biol. 2015 Mar 20;10(3):875-82. doi: 10.1021/cb500755j. Epub 2015 Jan 7.
Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase.
已知微生物对砷酸盐的抗性是由一类称为砷酸盐还原酶的特殊氧化还原酶赋予的。我们在添加了砷酸钠的培养基上对多拷贝基因进行了遗传筛选，这些基因能够使缺乏砷酸盐还原酶基因的大肠杆菌突变细胞（大肠杆菌ΔarsC）生长。我们发现谷胱甘肽S-转移酶B（GstB）的过表达弥补了ΔarsC等位基因的缺陷，并使细胞能够在含有高达5 mM砷酸钠的培养基上生长。有趣的是，与野生型大肠杆菌砷酸盐还原酶不同，通过GstB产生的砷酸盐抗性并不依赖于谷氧还蛋白提供的还原当量或催化性半胱氨酸残基。相反，发现两个精氨酸残基对于转移酶活性至关重要，这两个残基可能在GstB的亲电结合位点内配位砷酸盐底物。我们提供了生化证据，表明GstB以还原型谷胱甘肽（GSH）作为电子供体，直接将砷酸盐还原为亚砷酸盐。我们的结果揭示了细菌中砷酸盐解毒的一条途径，该途径取决于细菌谷胱甘肽S-转移酶以前未描述的功能。
