Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate
- Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)
- Department of Parasitology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)
- Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585 (Japan)
The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.
- OSTI ID:
- 22356220
- Journal Information:
- Acta Crystallographica. Section F, Vol. 61, Issue Pt 10; Other Information: PMCID: PMC1991314; PMID: 16511183; PUBLISHER-ID: vr5045; OAI: oai:pubmedcentral.nih.gov:1991314; Copyright (c) International Union of Crystallography 2005; Country of input: International Atomic Energy Agency (IAEA); ISSN 1744-3091
- Country of Publication:
- United Kingdom
- Language:
- English
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