Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products:: Atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction
Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products:: Atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction
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DOI：
10.1021/bi800413r
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发表时间：
2008-10-14
期刊：
影响因子：
2.9
通讯作者：
Harada, Shigeharu
中科院分区：
文献类型：
--
作者：
Inaoka, Daniel Ken;Sakamoto, Kimitoshi;Harada, Shigeharu
克氏锥虫二氢罗酸脱氢酶(DHOD)是DHOD家族中的一员，催化二氢罗酸氧化成二氢罗酸(第一半反应)，继而催化富马酸还原为丁二酸丁二酸酯(第二半反应)。二氢罗丹酸的氧化与FMN的还原是耦合的，还原的FMN在第二半反应中转化为富马酸丁二酸酯。已知TcDHOD对克氏锥虫的生存和生长是必不可少的，也是有效的药物靶标。乳酸乳球菌1A族DHOD的前半反应机理已被广泛研究，其基础是动力学同位素效应、诱变和X-射线结构测定的无配体形式和与前半反应产物甲酸根形成的络合物。在这份报告中，我们介绍了TcDHOD的晶体结构，包括无配体形式和带有抑制剂的络合物，生理底物以及第一和第二半反应的产物。这些配体都结合在TcDHOD的同一活性部位上，这与动力学研究所证明的1A族DHOD的单位乒乓式BI-BI机理是一致的。配体与TcDHOD的结合不会引起TcDHOD的任何结构变化，还原和氧化的FMN辅因子都是平面构象，这表明FMN辅因子被二氢盐还原生成阴离子还原的FMN。因此，它们应该是TcDHOD酶反应途径的良好模型，尽管在所确定的结构中，甲酸和富马酸通过氧化的FMN与TcDHOD结合，二氢甲酸与还原的FMN结合。CyS 130被确定为1ADHOD(Fagan，R.L.，Jensen，K.F.，Bjornberg，O.和Palfey，B.A.(2007)BioChemical 46,4028-4036)的活性中心碱基，它非常适合从二氢罗丹酸盐C5中提取质子并将其转移到外部水分子中。结合的富马酸为扭曲构象，导致部分电荷分离，表示为C-2(Delta-)和C-3(Delta+)。由于这种部分电荷分离，富马酸与还原的FMN的热力学有利的还原似乎以这样的方式进行：在TcDHOD中，C-2(Delta-)接受来自Cys130的质子，而C-3(Delta+)接受来自还原FMN N-5的氢化物(或氢化物当量)。
Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys 130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C-2(delta-) and C-3(delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C-2(delta-) accepts a proton from Cys 130 and C-3(delta+) a hydride (or a hydride equivalent) from reduced FMN N-5 in TcDHOD.