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Protein Science (2004), 13:1031-1042. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain

Majbritt Hansen1,2, Jérôme Le Nours1, Eva Johansson1,3, Torben Antal1,4, Alexandra Ullrich5, Monika Löffler5 and Sine Larsen1,3

1 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, DK-2100 Copenhagen, Denmark
2 Department of Clinical Microbiology, Copenhagen University Hospital, DK-2100 Copenhagen, Denmark
3 European Synchrotron Radiation Facility (ESRF), F-38043 Grenoble Cedex, France
4 Department of Clinical Biochemistry, Glostrup Hospital, DK-2600 Glostrup, Denmark
5 Institute for Physiological Chemistry, Philipps-University, D-35033 Marburg, Germany

(RECEIVED November 24, 2003; FINAL REVISION January 19, 2004; ACCEPTED January 20, 2004)



Abstract

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti-proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N-terminal extension, which folds into a separate domain comprising two {alpha}-helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N-terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 Å movement of the first residue in the first {alpha}-helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane-bound rat DHOD and membrane-associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.

Keywords: dihydroorotate dehydrogenase; inhibitor binding; brequinar; atovaquone; domain movement; membrane association

Abbreviations: DHOD, dihydroorotate dehydrogenase • DHO, dihydroorotate • DCIP, 2,3-dichlorophenolindophenol • HEPES, 4–(2-hydroxyethyl)-1-piperazineethanesulfonic acid • MPD, 2-methyl 2,4-pentanediol • TRIS, Tris-(hydroxymethyl)aminomethane, 2-amino-2–(hydroxymethyl)-1,3-propanediol • Q0, 2,3-dimethoxy-5-methyl-benzoquinone • DHODR, rat DHOD • DHODR-breq, structure of rat DHOD in complex with brequinar • DHODR-ato, structure of rat DHOD structure in complex with atovaquone • DHODH, human DHOD • DHODH-lefl, structure of human DHOD structure in complex with leflunomide • DHODH-breq, structure of human DHOD in complex with brequinar • DHODC, Escherichia coli DHOD • DHODA, Lactococcus lactis DHOD A • DHODB, Lactococcus lactis DHOD B • rmsd, root-mean-square deviation

Reprint requests to: Sine Larsen, Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark; e-mail: sine{at}ccs.ki.ku.dk or slarsen{at}esrf.fr; fax: 45-3532-0299 or 33-4-7688-2160.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03533004.


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