Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications

[Image: see text] Radical S-adenosyl-l-methionine (RS) enzymes operate on a variety of substrates and catalyze a wide range of complex radical-mediated transformations. Radical non-α-carbon thioether peptides (ranthipeptides) are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The RS enzyme PapB catalyzes the formation of thioether cross-links between Cys/Asp (or Cys/Glu) residues located in six Cys-X(3)-Asp/Glu motifs. In this report, using a minimal substrate that contains a single cross-link motif, we explore the substrate scope of the PapB and show that the enzyme is highly promiscuous and will accept a variety of Cys-X(n)-Asp sequences where n = 0–6. Moreover, we show that the enzyme will introduce in-line and nested thioether cross-links independently in peptide sequences that contain two motifs derived from the wild-type sequence. Additionally, the enzyme accepts peptides that contain d-amino acids at either the Cys or the Asp position. These observations are leveraged to produce a thioether cyclized analogue of the FDA-approved therapeutic agent octreotide, with a Cys-Glu cross-link replacing the disulfide that is found in the drug. These findings highlight the remarkable substrate tolerance of PapB and show the utility of RS RiPP maturases in biotechnological applications.