Carrier Protein‐Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC**

Abstract

The arylomycin antibiotics are potent inhibitors of bacterial type I signal peptidase. These lipohexapeptides contain a biaryl structural motif reminiscent of glycopeptide antibiotics. We herein describe the functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly. Unlike its enzymatic counterparts in glycopeptide biosynthesis, AryC converts free substrates without the requirement of any protein interaction partner, likely enabled by a strongly hydrophobic cavity at the surface of AryC pointing to the substrate tunnel. This activity enables chemo‐enzymatic assembly of arylomycin A2 that combines the advantages of liquid‐ and solid‐phase peptide synthesis with late‐stage enzymatic cross‐coupling. The reactivity of AryC is unprecedented in cytochrome P450‐mediated biaryl construction in non‐ribosomal peptides, in which peptidyl carrier protein (PCP)‐tethering so far was shown crucial both in vivo and in vitro.

Bacterial Cytochrome P450 Catalyzed Post‐translational Macrocyclization of Ribosomal Peptides**

“…P450s activate the substrates by hemolysis of a phenolic O−H or tryptophan indole N−H bond to form highly reactive radical intermediates [11] for oxidative coupling reactions. These oxidative coupling reactions were also observed in dimeric diketopiperazines (DKPs), such as naseseazines, [12] and non‐ribosomal peptides (NRPs), including glycopeptide antibiotic vancomycin [13] and lipopeptide arylomycin, [14] which expand the chemical space and complexity of macrocyclic peptides. This highlights the biocatalytic potential of P450 in peptide macrocyclization and opens new avenues for the discovery of new P450s and related peptides.…”

“…Indeed, P450s have been extensively explored for their potential as versatile biocatalysts, with significant efforts invested in discovery or engineering to enable access to more small molecules, [15] cyclic NPRs, and DKPs [12a,b,14,16] . Such P450‐catalyzed macrocyclization, however, either heavily limits to specific cyclodipeptides or requires a peptidyl carrier protein‐tethered precursor for substrate recognition, which complicates the utilization of these P450s and limits their biocatalytic potential.…”

P450-Modified Ribosomally Synthesized Peptides with Aromatic Cross-Links

“…HMBC correlations between H-9 and C-20, as well as H-19 and C-8, were observed, along with NOESY correlations between H-9 and H-19, revealing the C–C bond linkage between C-8 and C-20. Notably, 2 exhibited high structural similarities to the core structure of arylomycin A2, an antimicrobial lipopeptide generated via an NRPS pathway (Figure D). , This finding not only offers a basis for future chemoenzymatic synthesis of arylomycin but also highlights the potential of class C to produce a wide range of other cyclic tripeptides.…”

“…P450 enzymes have been shown to mediate cross-linking of some peptides, such as the cross-links of aromatic residues in nonribosomal peptides vancomycin and arylomycin. − In RiPPs, three P450 enzymes have been reported to be involved in the cross-links of aromatic amino acid residues, exemplified by cittilin, tryptorubin, and biarylitide (Figure B). − Compared with the complex and large NRPS biosynthetic gene clusters (BGCs), the BGCs for cittilin, tryptorubin, and biarylitide are much simpler, consisting of only one P450 gene and one precursor peptide gene (Figure C). We refer to these natural products as P450-modified RiPPs.…”

Non-Native Site-Selective Enzyme Catalysis

“…It was recently discovered that the enzyme AryC accomplishes a similar intramolecular C-C coupling reaction using only a lipophilic chain instead of a PCP tether to anchor the substrate within the enzyme active site. 366 Recently, a P450 from Streptomyces sp. MG-AR, originally identified for its ability to hydroxylate testosterone, was engineered to enable gram-scale oxidative coupling of the arylomycin core, demonstrating scalable production of this medicinally relevant motif (Scheme 55A).…”