METTL7A (TMT1A) and METTL7B (TMT1B) are responsible for alkyl S -thiol methyl transferase activity in liver .

S -Methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S- adenosyl-L-methionine dependent membrane-associated phase II enzyme known as thiol methyltransferase (TMT). TMT has a broad substrate specificity and methylates the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine pro-drugs, clopidogrel, and prasugrel. Despite TMT's role in the S- methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol-methyltransferase. METTL7B is an endoplasmic-reticulum-associated protein with similar biochemical properties and substrate specificity to TMT. Yet, the historic TMT inhibitor, 2,3-dichloro-α-methylbenzylamine (DCMB), does not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a thiol-methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and TMT, including inhibition by DCMB (IC 50 1.2 µM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which we have renamed TMT1A and TMT1B, respectively, that are responsible for TMT activity in human liver microsomes. Significance Statement We identified METTL7A (TMT1A) and METTL7B (TMT1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S- Methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity and identifying the enzymes responsible for this activity will improve our understanding of the DMPK properties of alkyl- or phenolic-thiol-containing therapeutics.