Evolutionary spread of protein L-(iso)aspartyl O -methyltransferases guides the discovery of distinct isoaspartate-containing peptides, pimtides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural products with a distinct biosynthetic logic, the enzymatic modification of genetically encoded precursor peptides. Although their structural and biosynthetic diversity remains largely underexplored, the identification of novel subclasses with unique structural motifs and biosynthetic pathways has been challenging. Here, we report that protein L-(iso)aspartyl O -methyltransferases (PIMTs) present in several RiPP subclasses are highly homologous. Importantly, we discovered that the apparent evolutionary transmission of the PIMT gene could serve as a basis to identify a novel RiPP subclass. Biochemical and structural analyses suggest that these homologous PIMTs commonly convert aspartate to isoaspartate via aspartyl- O -methyl ester and aspartimide intermediates, and often require cyclic or hairpin-like structures for modification. By conducting homology-based bioinformatic analysis of PIMTs, we identified over 2,800 biosynthetic gene clusters (BGCs) for known RiPP subclasses in which PIMTs install a secondary modification, and over 1,500 BGCs in which PIMTs function as a primary modification enzyme, thereby defining a new RiPP subclass, named pimtides. Our results suggest that the genome mining of proteins with secondary biosynthetic roles could be an effective strategy for discovering novel biosynthetic pathways of RiPPs.