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CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE
Waters, S. B., M Styblo, M. Beck, AND D J. Thomas. CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT S-ADENOSYL-1-METHIONINE: ARSENIC (III) METHYLTRANSFERASE. Presented at 5th Intern. Conf. on the Health Effects of Arsenic, San Diego, CA, July 15-19, 2002.
CLONING, EXPRESSION, AND MUTATIONAL ANALYSIS OF RAT
S-ADENOSYL-L-METHIONINE: ARSENIC(III) METHYLTRANSFERASE
Stephen B. Waters, Ph.D., Miroslav Styblo, Ph.D., Melinda A. Beck, Ph.D., University of North Carolina at Chapel Hill; David J. Thomas, Ph.D., U.S. Environmental Protection Agency
Methylation of inorganic arsenic (iAs) to yield methyl and dimethyl arsenic is a common feature of its metabolism in many species. In rat liver, a single enzyme, S-adenosyl-L-methionine: arsenic(III) methyltransferase (AsMT), catalyzes both methylation reactions. The predicted amino acid sequence of rat AsMT is similar to those of the predicted product of the cyt19 gene in the human and mouse genomes. The predicted sequences of the rat, mouse, and human proteins contain amino acid motifs commonly found in non-nucleic acid methyltransferases. Noteworthy features of these proteins are the presence of two methyltransferase motif II sequences (IIa & b) and C-terminal regions with conserved cysteines at three of the final eight residues. Rat cyt19 (rcyt19) and human cyt19 (hcyt19) have been cloned into expression vectors; both recombinant proteins methylate iAs, producing methyl- and dimethyl arsenic as minor and major metabolites, respectively. The rate of methylation of iAs is considerably greater for rcyt19 than for hcyt19. Differences in the rate of methylation of the rat and human enzyme may be due to single residue changes in motif IIa and IIb, or to the presence of a six amino acid insert in the hcyt19 sequence. Mutagenesis studies have focused on motif IIa, motif IIb and the cysteine-rich tail of rcyt19. Mutation of the conserved aspartate residue in motifs IIa (D185V) or IIb (D287V) abolishes methyltransferase activity. The I287M mutation in rcyt19 that alters the motif IIb sequence to that of hcyt19 reduces methyltransferase activity to 27.4% of wildtype rcyt19. Mutations C362S, C363S, and C369S reduce protein activity 3.0, 6.2 and 3.6 fold, respectively. Hence, the observed difference in the rate of methylation of iAs in human and rat hepatocytes could reflect between species differences in the amino acid sequence of cyt19 that affect its catalytic function. (This abstract does not necessarily reflect EPA policy.)
CORRESPONDING AUTHOR: David J. Thomas, Ph.D., National Health and Environmental Effects Research Laboratory, Office of Research and Development, MD74, U.S, Environmental Protection Agency, Research Triangle Park, NC 27711