Structure and mechanism for iterative amide N-methylation in the biosynthesis of channel-forming peptide cytotoxins

The polytheonamides are highly modified and potent, cytotoxic peptides with a unique D-helical structure (helical diameter similar to 4 angstrom) that affords selective membrane permeation of monovalent cations. Toxicity has been linked to promiscuous ion-channel behavior in studies of the prototypical polytheonamide B. Specific structural features of the beta-helical toxins include, among other modifications, C alpha-epirnerizations and N gamma-methylations, which have been highlighted as the early-stage modifications most critical for beta-helical formation. Here, we interrogate C alpha-epimerization and N gamma-methylation to understand the importance of these modifications for secondary structure. We characterize the mechanism of N gamma-methylations on the amide side chains of D-Asn, an enzymatic modification with little biochemical precedent. Crystal structures of the AerE methyltransferase in complex with its epimerized peptide substrate and S-adenosyl-homocysteine reveal features of substrate recognition and an unexpected metal-ion that may mediate methyl transfer to the poorly nucleophilic amide. These studies provide a framework for the engineering of novel beta-helical peptides with ion and membrane selectivity.