Biochemical Characterization of Drug Metabolizing Alkyl Thiol Methyltransferase 1A and 1B

Abstract

In humans, S-methylation of aromatic and alkyl thiols is catalyzed by thiopurine methyltransferase (TPMT), and a putative enzyme(s) thought to be responsible for alkyl thiol methyltransferase (TMT) activity. In this work, we identified METTL7A along with METTL7B as the enzymes responsible for TMT activity and renamed them thiol methyltransferase 1A and 1B (TMT1A and TMT1B), respectively. TMT1A and TMT1B exhibited many characteristics ascribed to historic TMT activity, including similar molecular weight, microsomal association, and broad substrate specificity for several alkyl thiol containing drugs, including spironolactone, captopril, and mertansine. Interestingly, TMT1A but not TMT1B is potently inhibited by a historic probe inhibitor of TMT activity, 2,3-dichloro-α-methylbenzylamine (DCMB). Utilizing DCMB to probe microsomal TMT activity suggests that TMT1A, not TMT1B, is responsible for most alkyl thiol methylation activity. To explain the difference in DCMB susceptibility, we compared the sequences of TMT1A and TMT1B and used homology models to identify key residues in the binding site. We then used site-directed-mutagenesis, to pinpoint a key tyrosine residue at position 47 (Tyr-47) in the binding site of TMT1A that was critical for DMCB inhibition. Mutating Tyr-47 to the corresponding Ser-47 in TMT1B abolished DCMB inhibition and confirmed the importance of Tyr-47 for DCMB binding and inhibition. Finally, we identified hydrogen sulfide (H2S) as a potential endogenous substrate of TMT1A and TMT1B and showed that the metabolite methyl sulfide (MeSH), similar to H2S, can efficiently adduct and sulfmethylate protein cysteines. To determine the physiological relevance of sulfmethylation, we developed a novel proteomics method to detect potential proteins adducted by MeSH. This protein adductomics method may help elucidate the endogenous role of MeSH in signaling and cell homeostasis.