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IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE
Citation:
XING, W., Z. DROBNA, M. STYBLO, B. ADAIR, AND D. J. THOMAS. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE. Presented at Society of Toxicology Annual Meeting, Charlotte, NC, March 25 - 29, 2007.
Description:
Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In human and rat, these are C32, C61, C85, C156, and C206. The role of the highly conserved residues in catalysis was tested by site-directed mutagenesis in which each C was changed to a serine (S) residue. Studies with rat AS3MT (rAS3MT) found that either C156S or C206S was catalytically inactive and that C61S preferentially catalyzed monomethylation of arsenic. In contrast, human AS3MT (hAS3MT) with C61S, C156S or C206S was catalytically active, suggesting hAS3MT compensated functionally for loss of C residues critical in rAS3MT. The hypothesis that other C residues in hAS3MT maintained activity in single mutants was tested using hAS3MT with double C to S mutants. Double mutants of conserved C residues (C61S/C156S, C61S/C206, C156S/C206S) were catalytically inactive. Double mutants of a conserved C and a non-conserved C (C156S/C334S, C206S/C334S) were inactive. Preservation of catalytic activity in single C to S mutants of hAS3MT and its loss in double C to S mutants suggests a non-conserved C (e.g., C334) in hAS3MT can provide redundancy that is lacking in rAS3MT. Redundant C residues in hAS3MT preserve catalysis by allowing formation of disulfide bonds needed for proper protein folding or by interactions with trivalent arsenicals which are substrates for this enzyme. (This abstract does not reflect US EPA policy.)