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Characterization of an Arsenate Reductase Encoded by ORF slr0946 from the Cyanobacterium Synechocystis SP. PCC6803
Author(s) -
Li R.,
Kennelly P. J.
Publication year - 2003
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.0022-3646.2003.03906001_102.x
Subject(s) - arsenate , arsenite , glutaredoxin , biochemistry , glutathione , biology , cysteine , enzyme , reductase , glutathione reductase , arsenic , chemistry , organic chemistry , glutathione peroxidase
Arsenic is a human carcinogen that is frequently encountered in high concentrations in both aqueous environments and in human drinking water supplies. Many microorganisms have evolved arsenical detoxification systems in which arsenate (AsV) is enzymatically reduced to arsenite (AsIII). In this study we report the expression and characterization of the first arsenate reductase from a cyanobacterium, the product of slr0946 from the cyanobacterium Synechocystis sp. PCC6803. Slr0946 was expressed as an C‐terminally ‘His‐tagged’ fusion protein. The recombinant protein efficiently catalyzed the reduction of arsenate to arsenite using reducing equivalents supplied from NADPH via glutathione and glutaredoxin. Thioredoxin, the traditional reductant for previously characterized ArsC's, was ineffective. Slr0946 displayed both relatively high catalytic efficiency, Vmax 3.1 μmol/min/mg, as compared to other arsenate reductases. Its Km for arsenate was 1.25 mM. Slr0946 also exhibited vestigial phosphohydrolase activity toward p‐nitrophenyl phosphate. Both arsenate reductase activity and phosphohydrolase activity were dependent upon the presence of the conserved cysteine residue, Cys8. Mutation of either of two closely paired cysteines in Slr0946, Cys80 or Cys82, but not Cys13 to serine eliminated arsenate reductase activity. However, each of these altered forms did catalyze the hydrolysis of pNPP, indicating that these cysteines were not essential to this catalytic function. It has further been suggested that glutathione forms a mixed disulfide with one of these cysteines during catalysis. This suggestion was at least partially supported by the demonstration that Slr0946 forms an SDS‐resistant complex with [35S]glutathione in a manner that is dependent on the presence of both Cys8 and arsenate.