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J. Biol. Chem., Vol. 277, Issue 49, 47476-47485, December 6, 2002
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§,
,,,,,,,§§,¶¶, and
From the 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.
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
Lehrstuhl für
Mikrobiologie/Biotechnologie,
Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle
28, 72076 Tübingen, Germany
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.
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