A Carbamoyl N-Methyltransferase Catalyzes N-Methylation of the Primary Amide in Ansacarbamitocin Biosynthesis

Primary amide-specific N-methyltransferases are extremely scarce in microbial secondary metabolism. Here, Asc-Orf2, an N-methyltransferase involved in the biosynthesis of ansacarbamitocins, was identified to catalyze the methylation of the 3-O-carbamoyl moiety. Structural analysis identified an unprecedented NPPH catalytic motif, offering a mechanistic basis to overcome the chemical inertness of primary amides. The 3-O-(N-methyl)-carbamoyl maytansinoid derivatives, modified via Asc-Orf2-catalyzed methylation, exhibited markedly enhanced antitumor activity, highlighting the magic methylation effect in bioactivity modulation. Furthermore, structure-targeted engineering expanded the catalytic scope of Asc-Orf2, enabling the directed synthesis of an N-allylated carbamoyl maytansinoid derivative optimized for antibody-drug conjugate payload.