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Originally published In Press as doi:10.1074/jbc.M206342200 on August 30, 2002

J. Biol. Chem., Vol. 277, Issue 49, 47476-47485, December 6, 2002
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Crystal Structure of OxyB, a Cytochrome P450 Implicated in an Oxidative Phenol Coupling Reaction during Vancomycin Biosynthesis*

Katja ZerbeDagger §, Olena Pylypenko§||, Francesca VitaliDagger , Weiwen ZhangDagger , Severine RousetDagger , Markus HeckDagger , Jan W. VrijbloedDagger , Daniel Bischoff**, Bojan Bister**, Roderich D. Süssmuth**, Stefan PelzerDagger Dagger , Wolfgang WohllebenDagger Dagger §§, John A. RobinsonDagger ¶¶, and Ilme Schlichting||||||

From the Dagger  Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland, the  Max Planck Institute for Molecular Physiology, Department of Physical Biochemistry, Otto Hahn Str.11, 44227 Dortmund, Germany, the || Max Planck Institute for Medical Research, Department of Biomolecular Mechanisms, Jahnstr. 29, 69120 Heidelberg, Germany, the ** Institut für Organische Chemie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany, and the Dagger Dagger  Lehrstuhl für Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany

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 Cys347 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.

* This work was supported in part by the Deutsche Forschungsgemeinschaft and European Union program "European Community- Access to Research Infrastructure Action of the Improving Human Potential Program to the EMBL Hamburg Outstation, Contract HPRI-CT-1999-00017."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AF486630.

The atomic coordinates and the structure factors (code 1lfk, 1lgf, and 1lg9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ Both authors contributed equally to this work.

§§ Supported by European Union MEGA-TOP Grant QLK3CT- 1990-00650.

¶¶ Supported by European Union Grant MEGA-TOP QLK3-CT-1990-00650 and Bundesamt für Bildung und Wissenschaft Grant 99.0241. To whom correspondence may be addressed: Organisch-chemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland. Tel.: 41-0-1-635-4242; Fax: 41-0-1-635-6833; E-mail: robinson@oci.unizh.ch.

|||| Supported by the "Richard und Anne-Liese Gielen-Leyendecker Stiftung and the Deutsche Forschungsgemeinschaft." To whom correspondence may be addressed: Max Planck Institute for Medical Research, Dept. of Biomolecular Mechanisms, Jahnstr. 29, 69120 Heidelberg, Germany. Tel.: 49-0-231-133-2738; Fax: 49-0-231-133-2797; E-mail: ilme.schlichting@mpimf-heidelberg.mpg.de.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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