Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cβ, and Cγ-Linked Thioether-Containing Peptides

Recently developed bioinformatic tools have bolstered the discovery of ribosomally synthesized and post-translationally modified peptides (RiPPs). Using an improved version of Rapid ORF Description and Evaluation Online (RODEO 2.0), a biosynthetic gene cluster mining algorithm, we bioinformatically mapped the sactipeptide RiPP class via the radical S-adenosylmethionine (SAM) enzymes that form the characteristic sactionine (sulfur-to-alpha carbon) cross-links between cysteine and acceptor residues. Hundreds of new sactipeptide biosynthetic gene clusters were uncovered, and a novel sactipeptide huazacin with growth-suppressive activity against Listeria monocytogenes was characterized. Bioinformatic analysis further suggested that a group of sactipeptide-like peptides heretofore referred to as six cysteines in forty-five residues (SCIFFs) might not be sactipeptides as previously thought. Indeed, the bioinformatically identified SCIFF peptide freyrasin was demonstrated to contain six thioethers linking the beta carbons of six aspartate residues. Another SCIFF, thermocellin, was shown to contain a thioether cross-linked to the gamma carbon of threonine. SCIFFs feature a different paradigm of non-alpha carbon thioether linkages, and they are exclusively formed by radical SAM enzymes, as opposed to the polar chemistry employed during lanthipeptide biosynthesis. Therefore, we propose the renaming of the SCIFF family as radical non-alpha thioether peptides (ranthipeptides) to better distinguish them from the sactipeptide and lanthipeptide RiPP classes.