Crystal Structure of OxyC, a Cytochrome P450 Implicated in an Oxidative C–C Coupling Reaction during Vancomycin Biosynthesis

Abstract

Gene inactivation studies point to the involvement of OxyC in catalyzing the last oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. Presently, the substrate and exact timing of the OxyC reaction are unknown. The substrate might be the bicyclic heptapeptide or a thioester derivative bound to a protein carrier domain. OxyC from the vancomycin producer Amycolatopsis orientalis was produced in Escherichia coli and crystallized, and its structure was determined to 1.9 Å resolution. OxyC gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. The addition of vancomycin aglycone to OxyC produced type I changes to the UV spectrum. OxyC exhibits the typical P450-fold, with the Cys ligand loop containing the signature sequence FGHGX-HXCLG and Cys-356 being the proximal axial thiolate ligand of the heme iron. The observation of a water molecule bound to the heme iron is consistent with the UV-visible spectra of OxyC indicating a low spin heme. A polyethylene glycol molecule occupying the active site might mimic the bicyclic heptapeptide substrate. Analysis of the structure of Oxy-proteins and other P450s indicates regions that might be involved in binding of the redox partner and possibly the protein carrier domain.Vancomycin and related glycopeptides are clinically important antibiotics that act as inhibitors of bacterial peptidoglycan biosynthesis (1, 2). These antibiotics typically possess a heptapeptide aglycone that is rigidified by side chain cross-linking involving several of its constituent aromatic amino acids (Fig. 1). As a result of this cross-linking, a molecular cavity is created within the glycopeptide core, the surface of which is complementary to that of the terminal D-Ala-D-Ala dipeptide unit present in intermediates of peptidoglycan biosynthesis (2). The glycopeptides are thus able to bind this dipeptide with high affinity. The aromatic cross-links arise formally through oxidative phenol coupling reactions. Although phenol coupling reactions are widespread in natural product biosynthesis as a whole, knowledge about the enzymes catalyzing such processes is sparse. It is clear, however, that members of the cytochrome P450 family have evolved that are able to promote specific phenol coupling reactions, both in glycopeptide antibiotic biosynthesis and in other areas of secondary metabolism, particularly alkaloid biosynthesis (3).Biosynthetic gene clusters for several glycopeptide antibiotics have been cloned and sequenced recently, including those for chloroeremomycin (4), balhimycin (5), and teicoplanin (6) production. The organization of the biosynthetic genes in each cluster is very similar. This includes in the chloroeremomycin cluster three large nonribosomal peptide synthetase (NRPS) 1 genes and three P450 genes (oxyA, oxyB, and oxyC), as well as other genes involved in the biosynthesis of the constituent amino acids...

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…An aryl-aryl coupling reaction is also reported for P450 158A2 that produces flaviolin dimer (26) and P450 OxyC that is involved in vancomycin biosynthesis (27). Based on the crystal structure of CYP158A2 complexed with the substrates, Zhao et al (26) proposed that the aryl-aryl coupling proceeds by means of direct hydrogen abstraction or direct bond formation with compound I.…”

Mechanisms of cytochrome P450 substrate oxidation: MiniReview

“…Such reactions are probably rare in the degradation of xenobiotics but are critical in pathways such as the synthesis of alkaloids [43] and antibiotics [45] (Figure 6). …”

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

“…OxyC catalyzes the formation of the C-C bond between the hydroxyphenylglycine at position 5 and the dihydroxyphenylglycine at position 7 in both vancomycin-and teicoplanin-like glycopeptides ( Fig. 1) (14). This enzyme is highly conserved in sequenced glycopeptide gene clusters and easily distinguished from related oxidative enzymes that appear in unrelated biosynthetic gene clusters, making it an ideal probe to use in the search for unique glycopeptide gene clusters.…”