Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis.

Peptide backbone α-N-methylations change the physicochemical properties of amide bonds to provide structural constraints and other favorable characteristics including biological membrane permeability to peptides. Borosin natural product pathways are the only known ribosomally encoded and posttranslationally modified peptides (RiPPs) pathways to incorporate backbone α-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting α-N-methyltransferase and separate precursor peptide substrate from the metal-respiring bacterium Shewanella oneidensis. A series of enzyme-precursor complexes reveal multiple conformational states for both α-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.