4″-Methyl-S-Adenosyl-l-Methionines Are Substrates for 4-Methylazetidinecarboxylic Acid Synthases

4-Methylazetidinecarboxylic acids (MeAZEs) are unusual nonproteinogenic amino acids found in nonribosomal peptides, such as bonnevillamide D and vioprolide A. The biosynthesis of MeAZEs is thought to proceed from S-adenosyl-l-methionine (SAM) via C-methylation and azetidine ring formation, but the order of these steps has remained unclear. Guided by our previous findings, we proposed that C4″-methylation of SAM by the cobalamin-dependent radical SAM enzyme BnvC, which is encoded in the biosynthetic gene cluster (BGC) of bonnevillamides, precedes azetidine ring formation. In this study, we identified the DUF364-containing enzyme Orf5, which is associated with the bnvC gene in the BGC of bonnevillamides, converts (4″R)-4″-methyl-SAM into cis-MeAZE. Further, we found that VioH, previously shown to catalyze azetidine ring formation from SAM in vioprolide A biosynthesis, transforms (4″S)-4″-methyl-SAM into trans-MeAZE. Structural modeling indicated that the C-methyl group was essential for efficient cyclization. These results elucidated the biosynthetic logic of MeAZEs and established C-methylation as a prerequisite for azetidine ring formation.