P450 Cyptide Synthase KwwB Catalyzes Trp-C5–Trp-N1 Cross-Linking and Accepts Diverse Precursor Peptides

Cytochrome P450 enzymes install C-C, C-N, or C-O cross-links on the precursor peptides of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we bioinformatically mapped P450 enzymes based on the known families. Through functional studies in <i>Escherichia coli</i>, the newly identified P450 cyptide synthase KwwB from <i>Kitasatospora</i> sp. GAS204B was found to catalyze the formation of a cross-link between Trp-C5 and Trp-N1 at the WxW motif. The substrate tolerance of the Trp-C5-Trp-N1 cross-linked P450 enzymes has not been determined. This work demonstrated that KwwB is a promiscuous enzyme capable of accepting a variety of substrates. This result provides an additional toolkit for cross-linked peptide modification.