Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase
Perturbing the Movement of Hydrogens to Delineate and Assign Events in the Reductive Activation and Turnover of Porcine Dihydropyrimidine Dehydrogenase
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扰动氢的运动来描述和分配猪二氢嘧啶脱氢酶的还原激活和周转事件
DOI：
10.1021/acs.biochem.1c00243
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发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
Moran, Graham R.
中科院分区：
文献类型：
--
作者：
Beaupre, Brett A.;Forouzesh, Dariush C.;Butrin, Arseniy;Liu, Dali;Moran, Graham R.
二氢嘧啶脱氢酶（DPD）的天然功能是利用NADPH产生的电子还原尿嘧啶和胸腺嘧啶的5,6-乙烯键。NADPH和嘧啶结合在由四个fe4s4中心桥接的由~ 60 Å隔开的不同活性位点上。我们已经证明，DPD经历了还原活化，从NADPH中获取了两个电子[Beaupre， b.a, et . (2020)Biochemistry 59, 2419-2431]。pH研究表明，周转率不受一般酸半胱氨酸671质子化状态的控制。C671变异体的活化分为两个阶段，特别是在低pH值下。描述的还原活化反应的光谱反褶积表明，初始相导致NADPH结合差谱中电荷转移吸收的积累。第二阶段的结果是两种黄素中的一种被还原。在低氧环境中，用NADPH和慢底物胸腺嘧啶浸泡C671S的x射线晶体结构分析，确定了FMN辅因子被还原的酶的活化形式。这些数据表明，电荷转移源于NADPH和FAD碱基的接近，随后的黄素是电子快速转移到FMN的结果，而没有FAD或fe4s4中心的还原形式的积累。这些数据表明，DPD的缓慢周转率是由携带C671残基的移动结构特征的运动所控制的。
The native function of dihydropyrimidine dehydrogenase (DPD) is to reduce the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons obtained from NADPH. NADPH and pyrimidines bind at separate active sites separated by ∼60 Å that are bridged by four Fe4S4centers. We have shown that DPD undergoes reductive activation, taking up two electrons from NADPH [Beaupre, B. A., et al. (2020)Biochemistry 59, 2419–2431]. pH studies indicate that the rate of turnover is not controlled by the protonation state of the general acid, cysteine 671. The activation of the C671 variants is delineated into two phases particularly at low pH values. Spectral deconvolution of the delineated reductive activation reaction reveals that the initial phase results in the accumulation of charge transfer absorption added to the binding difference spectrum for NADPH. The second phase results in reduction of one of the two flavins. X-ray crystal structure analysis of the C671S variant soaked with NADPH and the slow substrate, thymine, in a low-oxygen atmosphere resolved the presumed activated form of the enzyme that has the FMN cofactor reduced. These data reveal that charge transfer arises from the proximity of the NADPH and FAD bases and that the ensuing flavin is a result of rapid transfer of electrons to the FMN without accumulation of reduced forms of the FAD or Fe4S4centers. These data suggest that the slow rate of turnover of DPD is governed by the movement of a mobile structural feature that carries the C671 residue.