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.

Keywords: arylomycin; biaryl coupling; chemo-enzymatic synthesis; crystal structure; cytochrome P450.

Figure 1

Structures of vancomycin aglycon (1) and arylomycin A2 (2).

Scheme 1

A) Structure of the linear precursor peptide 3. Parts to be assembled by LPPS shown in green, by SPPS shown in pink. Dotted red line indicates the site of the strategic biocatalytic cross‐coupling reaction using AryC. B) LPPS synthesis of the lipopeptide side 11 b.

Scheme 2

Assembly of the linear arylomycin precursors 3 a/b.

Scheme 3

Top: Biocatalytic cross‐coupling of untethered, linear peptide precursors 3 a/b to arylomycin 2 a/b using AryC. Employed recombinant enzymes are depicted as colored spheres. Bottom: HPLC‐MS analyses of the coupling reaction of 3 b with different electron supply systems. Analyses of pure substrate (blue), reaction with Fpr/PetF (red) and Adx/AdR (green) are shown. m/z (substrate)=883.6. m/z (product)=881.7. For HRMS data and full HPLC‐MS chromatograms, see Supporting Information.

Figure 2

Structure of AryC. A) Overall folding topology of AryC displayed as ribbon. The heme and residues lining the active site are shown as stick model. Secondary structure elements are annotated as for P450s (α‐helices A−L, β‐sheets 1–3) and the flexible lid‐loop is indicated by a dashed line. B) Amino acid charges are mapped onto surface of AryC, highlighting the hydrophobic entrance channel to the active site. Proposed model for the binding‐mode of the HPG‐Ala‐Tyr‐moiety of the substrate (pink stick model) and likely positions of the lipophilic tail (green) are highlighted. The heme at the bottom of the active site is shown as cyan stick model.