Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (AsIII) produces organic arsenicals, including MMAIII, MMAV and DMAV with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated AsIII to DMAV as an end product and produced MMAIII and MMAV as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate AsIII as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated AsIII to MMAIII, MMAV and DMAV. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli. ► As3mt catalyzed biomethylation of AsIII to DMAV and produced toxic intermediates. ► As3mt activity is inhibited by elevated substrate concentrations and selenite. ► C160 and C165 are predicted as AsIII binding sites.