Padhi, C.; Nguyen, D. T.; Zhu, L.; Cha, L.; Wald, J. W.; Mitchell, D. A.; van der Donk, W.

Cytochrome P450s catalyze a diverse array of reactions including crosslinking of aromatic side chains in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). ApyO is a cytochrome P450 enzyme that forms a C-C bond between two tyrosines in a YLY motif in the substrate ApyA, the precursor peptide of the RiPP aminopyruvatide. We utilized cell-free translation to generate ApyA variants and probe the substrate tolerance of ApyO. Through Alphafold-based modelling and in vitro assays, we show that ApyO accepts the 10 C-terminal residues of ApyA and requires a conserved Arg/Lys in the substrate peptide. Inspired by substrate sequences found in orthologous biosynthetic gene clusters, we substituted one of the tyrosine residues with a tryptophan and observed that ApyO catalyzed the formation of an N-C bond between the indole of Trp and the C{epsilon}2 of Tyr. ApyO unexpectedly catalyzed formation of a C-O bond between the two tyrosine residues when we substituted the leucine residue in the YLY motif with tyrosine and tryptophan. We also show that a peptide containing a biaryl linkage and the C-terminal aminopyruvate displayed sub-nanomolar inhibitory activity against selected proteases. Overall, this study demonstrates plasticity in the manner of macrocyclization catalyzed by the P450 ApyO and provides a starting point for chemoenzymatic approaches towards producing diverse macrocyclic scaffolds.