RiPP enzyme heterocomplex structure-guided discovery of a bacterial borosin α-N-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 Shewanella oneidensis 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 S. oneidensis, probable boundaries for proteolytic maturation of the metabolite were projected and confirmed via comparison of S. oneidensis knockout and overexpression strains. All in all, the S. oneidensis 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.