CRYSTALLOGRAPHIC STUDY OF Escherichia coli FLAVOPROTEIN WrbA, A NEW NAD(P)H-DEPENDENT QUINONE OXIDOREDUCTASE

J. Wolfová^1,2, J. Brynda^1,3, J. R. Mesters⁴, J. Carey⁵, R. Grandori⁶, I. Kutá Smatanová^1,2

¹Institute of Physical Biology, University of South Bohemia České Budějovice, Zámek 136,
CZ-373 33 Nové Hrady, Czech Republic

²Institute of Systems Biology and Ecology, Academy of Science of the Czech Republic, Zámek 136,CZ-373 33 Nové Hrady, Czech Republic

³Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ-16637 Prague 6, Czech Republic

⁴Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany

⁵Chemistry Department, Princeton University, Washington Rd and William St, Princeton, NJ 08544-1009, USA

⁶Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy

julinka.w@tiscali.cz

The flavoprotein WrbA from Escherichia coli represents a new family of multimeric flavodoxin-like proteins implicated in cell protection against oxidative stress. The recently revealed NAD(P)H-dependent quinone oxidoreductase activity stimulated determination of crystal structures of E. coli WrbA and the following search for structural features characterising the new family of redox active proteins.

Well-formed tetragonal crystals of WrbA protein in complex with its cofactor flavin mononucleotide (FMN) were obtained using standard vapor-diffusion techniques. The diffracion data were collected for two different crystal forms and the X-ray crystal structures have been determined to a resolution of 2.0 Ǻ and 2.6 Ǻ.

Lower crystallizability observed previously for E. coli WrbA apoprotein (without bound FMN) indicates the positive influence of FMN on crystallization of WrbA protein. The suggested effect for FMN in favouring crystal lattice formation through the specific interaction with protein motivated further search for better crystals of WrbA apoprotein. Based on crystallization conditions found for WrbA-FMN complex the diffraction-quality crystals of WrbA apoprotein were obtained. Dependence of apoprotein crystal growth on temperature was observed. The crystals diffracted up to a resolution of 1.85 Ǻ.

Acknowledgements.

This work is supported by the Ministry of Education of the Czech Republic (projects: Kontakt ME640, MSM6007665808, LC06010) and by the Academy of Sciences of the Czech Republic (AV0Z60870520). We are grateful to the staff of EMBL/DESY in Hamburg for the support during data collection. Diffraction measurements at the synchrotron DESY/EMBL were supported of the European Community, Research Infrastructure Action under the FP6 “Structuring the European Research Area Specific Programme” to the EMBL Hamburg Outstation, Contract Number RII3-CT-2004-506008.