Conformational rearrangements enable iterative backbone N-methylation in RiPP biosynthesis

Borosins are ribosomally encoded and posttranslationally modified peptide (RiPP) natural products featuring amide-backbone alpha-N-methylation. Here, the authors report the discovery and characterization of type IV borosin 'split' pathways encoding distinct, separate alpha-N-methyltransferases and precursor peptide substrates. Peptide backbone alpha-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 alpha-N-methylations on translated peptides. Here we report the discovery of type IV borosin natural product pathways (termed 'split borosins'), featuring an iteratively acting alpha-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 alpha-N-methyltransferase and substrate. Along with mutational and kinetic analyses, our results give rare context into potential strategies for iterative maturation of RiPPs.