Biochemical and structural interrogation of a type IV borosin RiPP N-methyltransferase

Abstract

Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products often provide biosynthetic routes to unique peptide modifications not observed in primary metabolism. One such modification is α-N-methylation, which confers therapeutically useful properties to peptides that include membrane permeability, increased target specificity, and resistance to proteolytic degradation. In the past 7 years, α-N-methylation was discovered as a post-translational modification through elucidation of the omphalotin A biosynthetic pathway. This α-N-methyltransferase was the first of its kind, and with its discovery, founded a new class of RiPPs in fungi called borosins. Bioinformatic analyses uncovered a breadth of borosin gene clusters, revealing diverse protein architectural types, and > 2500 clusters throughout all domains of life. We selected a minimally iterative, type IV system, from the well-characterized organism Shewanella oneidensis MR-1, for in-depth mechanistic and structural characterization. Through this body of work, we are beginning to understand the "rules" for α-N-methylation in this model system. Moving forward, this work not only informs our understanding of this novel family of methyltransferases, but of iteratively acting RiPP biosynthetic enzymes in general.