Crystal Structure of OxyB, a Cytochrome P450 Implicated in an Oxidative Phenol Coupling Reaction during Vancomycin Biosynthesis

Abstract

Gene-inactivation studies point to the involvement of OxyB in catalyzing the first oxidative phenol coupling reaction during glycopeptide antibiotic biosynthesis. The oxyB gene has been cloned and sequenced from the vancomycin producer Amycolatopsis orientalis, and the hemoprotein has been produced in Escherichia coli, crystallized, and its structure determined to 1.7-Å resolution. OxyB gave UV-visible spectra characteristic of a P450-like hemoprotein in the low spin ferric state. After reduction to the ferrous state by dithionite or by spinach ferredoxin and ferredoxin reductase, the CO-ligated form gave a 450-nm peak in a UV-difference spectrum. Addition of putative heptapeptide substrates to resting OxyB produced type I changes to the UV spectrum, but no turnover was observed in the presence of ferredoxin and ferredoxin reductase, showing that either the peptides or the reduction system, or both, are insufficient to support a full catalytic cycle. OxyB exhibits the typical P450-fold, with helix L containing the signature sequence FGHGXHXCLG and Cys 347 being the proximal axial thiolate ligand of the heme iron. The structural similarity of OxyB is highest to P450nor, P450terp, CYP119, and P450eryF. In OxyB, the F and G helices are rotated out of the active site compared with P450nor, resulting in a much more open active site, consistent with the larger size of the presumed heptapeptide substrate.

Structural Characterization of OxyD, a Cytochrome P450 Involved in β-Hydroxytyrosine Formation in Vancomycin Biosynthesis

“…Additional examples are found in the biosynthesis of the vancomycin-type antibiotics: balhimycin (10), chloroeremomycin (11), and vancomycin, produced by different strains of Amycolatopsis ( Fig. 1) (12,13). In the biosyntheses of these compounds, the P450s OxyA, OxyB, and OxyC have been shown in vivo to be responsible for the oxidative phenolic coupling of the aromatic side chains found in the vancomycin aglycone (14 -18).…”

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

“…orientalis DSM40040 chromosomal DNA was the source of oxyC, as described earlier (7). The oxyC gene was amplified by PCR using the following primers (restriction sites are underlined): OxyCFor, 5Ј-GCTATCTAGACATATGGGTCACGATATCGATCAGGT-CG-3Ј; OxyCRev, 5Ј-TGATCAAGCTTAGGCCTTCACCAGGTAACC-GGTACCTGA-TCAGGGC-3Ј.…”

“…UV-visible Spectrophotometry-All spectra were obtained with a Varian Cary 3 double-beam spectrophotometer equipped with thermostated cell holder. CO difference spectra of reduced OxyC were measured as described earlier for OxyB (7). The imidazole complex with OxyC was generated by adding a buffered solution of imidazole to a final concentration of 50 mM to a solution of OxyC (2.5 M) in 50 mM Tris-HCl at pH 8.0.…”

“…So far, no reports of in vitro activity for the enzymes OxyA-C have been described. Recent attempts to demonstrate conversion of putative linear heptapeptide intermediates by OxyB failed to give significant amounts of any new cross-linked products resulting from oxidative coupling (7). Presently, the * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…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 (4). Using probes based on the vancomycin gtfE biosynthetic gene, a region homologous to three P450 genes (oxyA, oxyB, and oxyC) has also been cloned from the vancomycin producer Amycolatopsis orientalis (7). The involvement of the OxyA-C gene products in the three phenol coupling reactions required for balhimycin production, as well as the order of the coupling steps, has been established by gene knock-out experiments (8,9).…”