RiPP enzyme heterocomplex structure-guided discovery of a bacterial borosin α--methylated peptide natural product.

Amide peptide backbone methylation is a characteristic post-translational modification found in a family of ribosomally synthesized and post-translationally modified peptide natural products (RiPPs) called borosins. Previously, we bioinformatically identified >1500 putative borosin pathways in bacteria; however, none of the pathways were associated with a known secondary metabolite. Through in-depth characterization of a borosin pathway in MR-1, we have now identified a bacterially derived borosin natural product named Shewanellamide A. Borosin identification was facilitated by the creation and analysis of a series of precursor variants and crystallographic interrogation of variant precursor and methyltransferase complexes. Along with assaying two proteases from , probable boundaries for proteolytic maturation of the metabolite were projected and confirmed comparison of knockout and overexpression strains. All in all, the natural product was found to be a 16-mer linear peptide featuring two backbone methylations, establishing Shewanellamide A as one of the few borosin metabolites yet identified, and the first from bacteria.