P450 Cyptide Synthase AscB Catalyzes Fused Cyclophane at the YxWxH Motif on the Precursor Peptides

Cyptides are a new class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with the unique feature of biaryl C-C, C-N, or C-O cross-links formed by P450 cyptide synthases (P450s). Through functional studies in Escherichia coli, we discovered two newly identified P450 enzymes, AscB from Amycolatopsis sacchari DSM 44468 and MmsB from Micromonospora sp. HM5-17, that can catalyze the cross-linking between Tyr-C3 and Trp-N1, as well as the hydroxylation at Trp-C5 in the YxWxH motif. However, only AscB was able to catalyze the formation of a second cyclophane between Trp-C4 and His-Nτ, generating a fused cyclophane in the YxWxH motif. This is the first report of an enzyme that can catalyze such cross-linking in RiPPs.