LAUSR

N6‐methyladenosine (m6A), widely distributed in both coding and noncoding RNAs, regulates the epigenetic signals and RNA metabolism in eukaryotes. Although this posttranscriptional modification is frequently observed in messenger and ribosomal RNA, it is relatively rare in transfer RNA. In Escherichia coli, TrmM encoded by yfiC is the tRNA‐specific N6 methyltransferase, which modifies the A37 residue of tRNAVal (cmo5UAC) using S‐adenosyl‐l‐methionine as a methyl donor. However, the structure–function relationship of this enzyme is not completely understood. In this report, we determined two x‐ray crystal structures of Mycoplasma capricolum TrmM with and without S‐adenosyl‐l‐homocysteine, which is a reaction product. We also demonstrated the cellular and in vitro activities of this enzyme in the m6A modification of tRNA and the requirement of a divalent metal ion for its function, which is unprecedented in other RNA N6 methyltransferases, including the E. coli TrmM. Our results reveal that the dimeric form of M. capricolum TrmM is important for efficient tRNA binding and catalysis, thereby offering insights into the distinct substrate specificity of the monomeric E. coli homolog.