Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with 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 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.
Inaoka
Daniel Ken
DK
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
Takashima
Eizo
E
Osanai
Arihiro
A
Shimizu
Hironari
H
Nara
Takeshi
T
Aoki
Takashi
T
Harada
Shigeharu
S
Kita
Kiyoshi
K
D050960:Aminomethyltransferase / Q000737:chemistry; D000818:Animals; D003460:Crystallization; D000090722:Dihydroorotate Dehydrogenase; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009963:Orotic Acid / Q000737:chemistry*; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D011743:Pyrimidines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013385:Succinate Dehydrogenase / Q000737:chemistry; D013696:Temperature; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014961:X-Ray Diffraction
D000090722:Dihydroorotate Dehydrogenase; D011743:Pyrimidines; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013385:Succinate Dehydrogenase; D050960:Aminomethyltransferase