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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 A 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.
Highlights
Vancomycin and related glycopeptides are clinically important antibiotics that act as inhibitors of bacterial peptidoglycan biosynthesis [1, 2]
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, 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 has been cloned from the vancomycin producer Amycolatopsis orientalis [7]
Summary
Optimal substrate for OxyC is unknown, but it should be a doubly cross-linked heptapeptide either in a free form or possibly linked as a thioester to a PCD. This thioester, after transformation on OxyC and hydrolytic cleavage of the Cterminal thioester, would afford vancomycin aglycone. Nonchlorinated cross-linked intermediates have been isolated from gene knock-out mutants of the balhimycin producer [8], showing that the chlorine atoms are not essential for crosscoupling to occur in this pathway. The potential substrates, a doubly cross-linked free heptapeptide or one bound to a PCD, are so far not available to test enzymatic activity. The crystal structure of OxyC does reveal a well ordered polyethylene glycol (PEG) molecule bound in the presumed active site of the enzyme
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