Pathway of Human AS3MT Arsenic Methylation

Abstract

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396–416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183–191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates.

Arsenic Methylation and Volatilization by Arsenite S -Adenosylmethionine Methyltransferase in Pseudomonas alcaligenes NBRC14159

“…PaArsM contains four fully conserved cysteines at the residues 24, 52, 145, and 195 (indicated by triangles in Fig. 2), which are characteristic of all ArsM proteins investigated to date and are probably involved in As(III) binding (23,36,37). Three sequence motifs possibly involved in the interactions with S-adenosylmethionine (SAM) are underlined Fig.…”

“…Several schemes have been proposed to explain the process of As biomethylation (13,23,38,39) via sequential methylation steps producing mono-, di-, and trimethylated species. The main product of the PaArsM protein activity was found to be DMAs(V) in both P. alcaligenes and E. coli expressing PaarsM.…”

“…ArsM has also been studied in a number of eukaryotic organisms, including the human and several algal species (20)(21)(22). In the amino acid sequences of ArsM, four cysteine residues are highly conserved in different organisms and are likely to be involved in the catalytic function (23). Genes encoding ArsM have also been identified and sequenced in several microorganisms, showing certain genetic organization.…”

Arsenic methylation by an arsenite S-adenosylmethionine methyltransferase from Spirulina platensis

“…N-or C-terminal residues of enzymes related to arsenic resistance are usually not required for their functions, while cysteine residues are often conserved and play an important role in the structure and function of the enzymes (Dheeman et al, 2014;Fomenko et al, 2008;Marapakala et al, 2012). The multiple-sequence alignment of ArsM illustrates that three cysteine residues are highly conserved and were shown to be involved in As(III) binding and catalysis of As methylation by site-directed mutagenesis (Ajees et al, 2012;Dheeman et al, 2014;Marapakala et al, 2012Marapakala et al, , 2015.…”

“…The multiple-sequence alignment of ArsM illustrates that three cysteine residues are highly conserved and were shown to be involved in As(III) binding and catalysis of As methylation by site-directed mutagenesis (Ajees et al, 2012;Dheeman et al, 2014;Marapakala et al, 2012Marapakala et al, , 2015. Herein, we constructed a C-terminal truncated SpArsM and three cysteine mutants (C59S, C186S, C238S) to investigate whether deleting or mutating these residues had an effect on the function of SpArsM.…”