Carrier Protein-Free Chemo-Enzymatic Synthesis of Arylomycin A2 and Structural Characterization of the Cytochrome P450 AryC

Abstract

Most antibiotics in clinical use are inspired by natural products. Prominent examples are the glycopeptides, such as vancomycin. These compounds contain biaryl-and biaryl-ether bonds that are crucial for biological activity and biosynthetically get installed by dedicated cytochrome P450 enzymes. The application of these biocatalysts in the chemo-enzymatic synthesis of novel glycopeptides has been impeded by their strict requirement for peptidyl carrier-protein (PCP) bound substrates and additional X domain interactions. This necessitates equimolar amounts of protein tethers, precluding truly catalytic applications. We describe the functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly, and its application in the chemo-enzymatic synthesis of arylomycin A2. AryC efficiently 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. The resulting reactivity of AryC is unprecedented in cytochrome P450-mediated biaryl construction in non-ribosomal peptides, in which PCP-tethering so far was crucial both in vivo and in vitro. Our work thus provides a basis for the development of general biocatalytic platforms for the efficient biocatalytic synthesis of biaryl peptide antibiotics.

Bacterial Cytochrome P450-catalyzed Post-translational Macrocyclization

“…These conserved aromatic residues of core peptide allow P450 enzymes to activate substrate via homolysis of phenolic O−H bond or tryptophan indole N−H bond to form highly reactive radical intermediates 15 , which can undergo diverse coupling reactions to forge C-C, C-O, or C-N bonds. These oxidative couplings were also observed in cyclodipeptides (CDPs), such as diketopiperazine 16 , and non-ribosomal peptides (NRPs), including glycopeptide antibiotics vancomycin 17 and lipopeptide arylomycin 18 , expanding the chemical space and complexity of macrocyclic peptides and highlighting the biocatalytic potential of P450 in peptide macrocyclization.…”

“…Significant efforts have been devoted to P450 discovery or engineering to establish a biocatalytic entry into these bioactive NPRs or CDPs 16,[18][19][20][21][22][23] . Such 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 P450 enzymes and limits the enzyme engineering.…”

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. 368 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).…”

Structural and Mechanistic Insights into Oxidative Biaryl Coupling to form Arylomycin Core by an Engineered CYP450

“…For instance, in the arylomycin biosynthetic gene cluster, a specific cytochrome P450 protein, encoded by the aryC gene (named as Wild type 1), is implicated in the formation of crucial biaryl carbon-carbon bonds. 24,25 However, no study has been done about the active site or neighbouring site transformations that enable a monooxygenase to catalyse C-C bond forming reaction. Therefore, it is quite important to address such issue which will be helpful to synthesise more natural products like arylomycin by engineering the P450 enzyme.…”