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BiochemistryAngewandte Chemie International Edition
Peer-reviewed journal article

Substrate-dependent crosslinking by the cytochrome P450 from aminopyruvatide biosynthesis

Chandrashekhar PadhiiD, Dinh Thanh NguyeniD, Lingyang ZhuiD, Lide ChaiD, Jesse W WaldiD, Douglas A. MitchelliD, Wilfred A. van der DonkiD

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 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 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.

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In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 3 (1 Sept 2026) · CC BY 4.0

The P450 ApyO crosslinks a 10-residue C-terminal peptide, and substitutions in the YLY motif switch the linkage among C−C, N−C and C−O bonds.

P450-installed crosslinks in RiPPs (ribosomally synthesized and post-translationally modified peptides) vary in bond type, and mass spectrometry alone cannot assign them. Because ApyO acts on unmodified precursor, its substrate scope can be mapped directly, showing that residues beside the crosslinked tyrosines can redirect the chemistry.

Key results

  1. ShownA C-terminal 10-residue peptide of ApyA is a sufficient ApyO substrate in cell-free translation, while an 8-residue truncation gives no product. Kinetics were measured on a synthetic acetylated 10-mer.

    ApyAct (fMRMGEGYLYD) ~70% conversion by MALDI-ToF and LC-MS; 8-mer fMGEGYLYD gave no product; Ac-ApyAct kcat 1.63 ± 0.04 min^−1, Km 80 ± 7 µM. · AlphaFold-based prediction of substrate binding

  2. ShownArg1 is essentially indispensable for crosslinking, and Glu4 contributes. This matches the AlphaFold3 prediction of an Arg1 hydrophilic pocket and a Glu4 to ApyO Arg47 contact.

    All 19 Arg1 replacements were deleterious; R1K only ~8% conversion (WT ~70%). E4A ~56% conversion; E4K ~23%. Conserved Arg/Lys at position 1: 74% Arg, 22% Lys among precursors. · AlphaFold-based prediction of substrate binding; Site directed mutagenesis of ApyA

  3. ShownThe Y6W variant is crosslinked through an N−C bond between the Trp6 indole nitrogen and Cε2 of Tyr8, instead of the native Tyr−Tyr C−C bond. This was assigned by NMR of the GluC-released pentapeptide.

    2 Da mass loss for Y6W and Y8W by LC-MS; HMBC cross peak Hδ1 of Trp6 (7.01 ppm) to Cε2 of Tyr8 (129.8 ppm); NOESY cross peaks between Trp6 and Tyr8 protons. WLY occurs in n = 23 vs YLW in n = 2 orthologs. · Ortholog-guided mutation of apyA; Structural characterization of ApyO-modified…

  4. ShownReplacing the central Leu7 with Tyr or Trp gives two constitutional isomers. Isomer 1 keeps the native Cε2−Cε2 C−C crosslink; isomer 2 has a non-native C−O bond from Tyr6 Cε2 to the Tyr8 phenolic oxygen.

    CFE conversion: L7Y near quantitative, L7I ~60%, L7V ~57%, L7W ~39%, L7F ~8%, L7T ~3%, L7G ~1%. Two isobaric products for L7Y, L7W, L7F, L7I, L7V; NMR assigned only L7Y and L7W isomers; L7T and L7G gave one peak. · Site directed mutagenesis of ApyA; Identification of two isobaric products from…; Structural characterization of L7Y and L7W…

  5. SuggestedDesmethylaminopyruvatide A, which carries the α-keto-β-amino acid, inhibits papain-family Cys proteases at sub-nanomolar potency. ApyO-only crosslinked peptides were only modestly active, suggesting the keto acid is critical.

    IC50 0.6 nM cathepsin L, 0.5 nM cathepsin B, 0.3 nM papain; no inhibition of trypsin, chymotrypsin or Mpro. ApyO-only peptides at 25 µM: L7Y isomer-2 inhibited ACE (IC50 1.1 µM) and papain (11.7 µM); L7W isomer-2 weakly inhibited ACE (~40 %). · Bioactivity

Methods, data and reproducibility

Cell-free translation of ApyA C-terminal variants was reacted with purified ApyO and redox partners and read out by LC-HRMS, guided by AlphaFold3 modelling. Crosslinks were assigned by NMR of GluC-released pentapeptides from full-length co-expression, followed by protease inhibition assays.

  • PHI-BLAST search with ApyA and YLY pattern, giving nonredundant putative precursor peptides in aminopyruvatide-like BGCs · bacteria (ApyA from B. thailandensis E264) · n = 53 nonredundant sequences · ABC34935.1 (ApyA); ABC35200.1 (ApyO)
  • Cell-free translated ApyAct 10-mer variants (Arg1, Glu4, Tyr6, Leu7, Tyr8 substitutions) reacted with ApyO
  • Full-length His-tagged ApyA variants (Y6W, Y8W, L7Y, L7W) co-expressed with ApyO in E. coli (BL21 (DE3) TUNER for L7Y and L7W), GluC-digested for MS; NMR on Y6W, L7Y, L7W · Escherichia coli (host)
  • Protease inhibition panel on GluC-digested ApyO products and desmethylaminopyruvatide A (cathepsins B and L, papain, trypsin, chymotrypsin, ACE, Mpro)
  • Deposited data for the study (Mendeley Data) · doi:10.17632/cr798k4bhw.1
Methods & tools
AlphaFold3, PHI-BLAST, RODEO, PURExpress cell-free translation, LC-HRMS and MS/MS, MALDI-ToF MS, NMR (TOCSY, NOESY, HSQC, HMBC), GluC endopeptidase digestion, Protease inhibition IC50 assays
Reproducibility gaps
No code repository is given. Data are deposited on Mendeley Data (DOI only), and experimental procedures, MS and NMR spectra and assignment tables are in the Supporting Information, not the main text.
Limitations (5)
  • The origin of the different crosslinking outcomes (C−C vs C−O) is unclear; it probably reflects subtle changes in the spatial arrangement of the two Tyr residues, and mechanistic studies are still needed. · stated by the authors
  • The truncated formyl-Met peptide only imperfectly mimics full-length ApyA: for L7Y the CFE favored one isomer more than co-expression did, though the key trends were recapitulated. · stated by the authors
  • The L7Y product with an α-keto-β-amino acid could not be obtained in useful yield (inefficient conversion in E. coli and in Burkholderia), so protease inhibition by such a non-native C−O isomer was not tested. Effect of the β-methyl group was also not determined. · stated by the authors
  • Only Y6W, L7Y and L7W products were structurally assigned by NMR. Other variants giving two isobaric products (L7F, L7I, L7V) and the Y8H crosslinked product were not assigned, so their bond types are inferred. · noted in this summary
  • Conversion values are approximate single-condition LC-MS estimates; no replicate counts or variance are reported, and the AlphaFold3 contacts were tested only indirectly by mutagenesis. · noted in this summary

Who should read:Read this if you work on P450 RiPP crosslinking enzymes, want to see how substrate edits redirect biaryl, N−C or C−O bond formation, or are screening macrocyclic protease inhibitors.

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