Many pharmacologically important peptides are bacterial or fungal in origin and contain nonproteinogenic amino acid (NPA) building blocks. Recently, it was reported that, in bacteria, a cyclopropane-containing NPA 1-aminocyclopropanecarboxylic acid (ACC) is produced from the L-methionine moiety of S-adenosyl-L-methionine (SAM) by non-canonical ACC-forming enzymes. On the other hand, it has been suggested that a monomethylated ACC analogue, 2-methyl-ACC (MeACC), is derived from L-valine. Therefore, we have investigated the MeACC biosynthesis by identifying a gene cluster containing bacterial MeACC synthase genes. In this gene cluster, we identified two genes, orf29 and orf30, which encode a cobalamin (B12)-dependent radical SAM methyltransferase and a bacterial ACC synthase, respectively, and were found to be involved in the MeACC biosynthesis. In vitro analysis using their recombinant enzymes (rOrf29 and rOrf30) further revealed that the ACC structure of MeACC was derived from the L-methionine moiety of SAM, rather than L-valine. In addition, rOrf29 was found to catalyze the C-methylation of the L-methionine moiety of SAM. The resulting methylated derivative of SAM was then converted into MeACC by rOrf30. Thus, we demonstrate that C-methylation of SAM occurs prior to cyclopropanation in the biosynthesis of a bacterial MeACC (norcoronamic acid).