# Substrate-Dependent Crosslinking by the Cytochrome P450 From Aminopyruvatide Biosynthesis

Article · Padhi C, Nguyen DT, Zhu L, Cha L, Wald JW, Mitchell DA, van der Donk WA

- Archive id: doi:10.1002/anie.7714055
- Published: 2026-09-15
- Venue: Angewandte Chemie (International ed. in English)
- DOI: https://doi.org/10.1002/anie.7714055
- PMID: 42745189
- PMCID: PMC13703928
- Citations: 0
- Source URL: https://doi.org/10.1002/anie.7714055
- PDF: https://europepmc.org/articles/PMC13703928/pdf
- Provenance: europepmc

## 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.

_Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._

Topics: [AI Protein Design & Structure Prediction](https://space-kitty.com/topics/protein-design)

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