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J. Biol. Chem., Vol. 278, Issue 47, 46727-46733, November 21, 2003

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

Olena Pylypenko, Francesca Vitali¶, Katja Zerbe¶, John A. Robinson¶||, and Ilme Schlichting**

From the Department of Physical Biochemistry, Max Planck Institute for Molecular Physiology, Otto Hahn Strasse 11, 44227 Dortmund, Germany, Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany, and ^¶Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

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.

Received for publication, June 18, 2003 , and in revised form, July 25, 2003.

The atomic coordinates and structure factors (code 1UED) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence may be addressed: Organisch-chemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland. Tel.: 41-1-635-4242; Fax: 41-1-635-6833. ** To whom correspondence may be addressed: Dept. of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany. Tel.: 49-231-133-2738; Fax: 49-231-133-2797; E-mail: ilme.schlichting{at}mpimf-heidelberg.mpg.de.

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