Substrate‐Dependent Crosslinking by the Cytochrome P450 From Aminopyruvatide Biosynthesis

ABSTRACT Cytochrome P450s catalyze an 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 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. Inspired by substrate sequences in orthologous biosynthetic gene clusters, we substituted one of the tyrosine residues with a tryptophan and observed that ApyO catalyzed formation of an N─C bond between the indole of Trp and Cε2 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 or tryptophan. A peptide containing a biaryl linkage and C‐terminal aminopyruvate displayed sub‐nanomolar inhibition of select proteases, with the aminopyruvate group critical for activity. Overall, this study demonstrates plasticity in the manner of macrocyclization catalyzed by the P450 ApyO.