Biosynthesis of Methylazetidinecarboxylic Acid with Opposite Stereoconfigurations in Vioprolides and Bonnevillamides

Vioprolides and bonnevillamides are both peptide natural products with antibacterial and anti-inflammatory activities. They both contain a 4-methylazetidine-2-carboxylic acid (MAZ) moiety with opposite stereoconfigurations of the methyl groups. The biosynthesis of MAZ has been proposed to involve the methylation of the precursor azetidine-2-carboxylic acid (AZE). Herein, we demonstrate that the biosynthesis of the MAZ moiety in vioprolides and bonnevillamides involves two cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferases (B12-RSMTs) VioG and BnvC, which catalyze the stereoselective methylation of the C-4″ position of SAM rather than AZE to generate 4″-methyl-SAM (4″-Me-SAM). We further demonstrated that the previously proposed SAM cyclase VioH prefers 4″-Me-SAM rather than SAM as the substrate. VioH selectively cyclizes (4″S)-4″-Me-SAM, whereas BnvI cyclizes (4″R)-4″-Me-SAM to the corresponding MAZ moiety. Therefore, we demonstrate that the SAM methylation step occurs prior to cyclization during MAZ biosynthesis and reveal strategies for generating different configurations of methyl-azetidine in natural products. VioG and BnvC are two B12-RSMTs that methylate the same substrate with opposite stereoconfigurations.