Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate
Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate
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DOI：
10.1107/s174430910502659x
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发表时间：
2005-10-01
影响因子：
0.9
通讯作者：
Kita, K
中科院分区：
文献类型：
--
作者：
Inaoka, DK;Takashima, E;Kita, K
二氢乳清酸脱氢酶（DHOD）催化二氢乳清酸氧化为乳清酸，这是嘧啶从头生物合成的第四步，也是唯一的氧化还原反应。来自克氏锥虫的DHOD（TcDHOD）已在大肠杆菌中表达为重组蛋白并纯化至均一。TcDHOD乳清酸盐配合物的晶体生长在277 K的坐滴气相扩散技术，使用聚乙二醇3350作为沉淀剂。使用同步加速器辐射（λ = 0.900埃），晶体的分辨率优于1.8埃。在100 K下收集X射线衍射数据，并处理至1.9 A分辨率，完整性为98.2%，总体R合并为7.8%。TcDHOD晶体属正交晶系，空间群为P2（1）2（1）2（1），晶胞参数a = 67.87，B = 71.89，c = 123.27埃。在不对称单元中存在两个分子（2 x 34 kDa），得到每蛋白质重量的晶体体积（V-M）为2.2埃（3）Da（-1），溶剂含量为44%。
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 angstrom resolution using synchrotron radiation (lambda = 0.900 angstrom). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall R-merge 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 angstrom. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (V-M) of 2.2 angstrom(3) Da(-1) and a solvent content of 44%.