P450 cyptide synthase KwwB catalyzes non-native cross-link at Tyr-C3-Trp-N1 and tolerates leader peptide mutations.

Cyptides refer to a rapidly growing class of ribosomally synthesized and post-translationally modified peptides (RiPPs) containing biaryl cyclophanes installed by P450 enzymes. Here, we further investigated P450 cyptide synthase KwwB from Kitasatospora sp. GAS204B, which expands the KwwB-catalyzed chemical repertoire to non-native cross-link between Tyr-C3 and Trp-N1 at the YxW motif. In addition, we demonstrated that KwwB tolerates leader peptide mutations at positions -11 through -2, with the exception of position -4, which contains a conserved Pro residue. This result provides an additional toolkit for cross-linked peptide modification.