Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase
Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase
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DOI：
10.1529/biophysj.107.120055
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发表时间：
2008-03-01
影响因子：
3.4
通讯作者：
Costa-Filho, Antonio J.
中科院分区：
文献类型：
--
作者：
Couto, Sheila G.;Nonato, M. Cristina;Costa-Filho, Antonio J.
二氢乳清酸脱氢酶（DHODH）在嘧啶从头合成途径的第四步期间催化二氢乳清酸氧化成乳清酸。在快速增殖的哺乳动物细胞中，嘧啶补救途径不足以克服核苷酸合成途径中的缺陷。此外，由于某些寄生虫缺乏补救酶，仅依赖于从头途径，DHODH抑制已被证明是阻断嘧啶生物合成的有效方式。大肠杆菌DHODH（EcDHODH）是2类DHODH，发现通过N-末端延伸与细胞溶质膜缔合。我们用电子自旋共振（ESR）研究了EcDHODH与1，2-二油酰-sn-甘油-磷脂酰胆碱/去污剂囊泡的相互作用。通过位于磷脂衍生物不同位置的自旋标记物监测由酶诱导的囊泡动态结构的变化。在EcDHODH的存在下，获得标记5-和10-磷脂酰胆碱的双组分ESR光谱，而其他探针显示单组分光谱。具有与探针的快速运动制度相关的特征的额外光谱分量的出现归因于膜疏水区域中的缺陷样结构的形成。这可能是蛋白质在催化过程中捕获用作电子受体的醌的机制。使用特定的光谱模拟程序，使我们能够表征的ESR光谱的极性和流动性的变化，周围的自旋标记的磷脂。我们相信这是第一份关于2类DHODH与膜系统结合的直接证据的报告。
Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 1 0-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.