Interaction of benzoate pyrimidine analogues with class 1A dihydroorotate dehydrogenase from Lactococcus lactis.
Interaction of benzoate pyrimidine analogues with class 1A dihydroorotate dehydrogenase from Lactococcus lactis.
复制标题
苯甲酸酯嘧啶类似物与乳酸乳球菌的 1A 类二氢乳清酸脱氢酶的相互作用。
DOI：
10.1021/bi7001554
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Palfey,BruceA
中科院分区：
文献类型：
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作者：
Wolfe,AbigailE;Thymark,Majbritt;Gattis,SamuelG;Fagan,RebeccaL;Hu,Yu-chen;Johansson,Eva;Arent,Susan;Larsen,Sine;Palfey,BruceA
二氢羊角酸脱氢酶（DHODs）催化二氢羊角酸氧化为羊角酸，是嘧啶生物合成中唯一的氧化还原反应。在所有结构表征的DHODs中，嘧啶结合位点非常相似，这表明鉴定针对该位点的类特异性抑制剂的前景很差。尽管如此，已经鉴定出两种与乳酸乳球菌1A类DHOD特异性结合的化合物，3,4-二羟基苯甲酸酯（3,4- diohb）和3,5-二羟基苯甲酸酯（3,5- diohb） [Palfey等人（2001）J]。中华医学杂志，2014,28(4):661 ~ 664。抑制剂与1A类DHOD的结合机制。现在已经对乳酸乳酸进行了详细的研究，并在这里进行了报道。滴定结果表明，3,4- diohb在较高的pH下结合更紧密，而3,5- diohb则相反。等温滴定量热法和吸光度法表明，3,4- diohb与酶结合后电离成酚酸盐，而3,5- diohb则没有。在3,4-二ohb络合物中形成的电荷转移带允许在停流实验中观察到结合动力学。从pH值6到pH值8，结合速度足够慢，并且（至少）是一个两步过程，包括快速形成一个异构化到最终的电荷转移络合物的络合物。Orotate和3,5- diohb结合太快，无法直接跟踪，但它们的解离动力学通过竞争研究，并充分描述了一个步骤。测定了两种抑制剂配合物的晶体结构，表明3,5-二ohb的结合方向与旋合方向相同。相反，3,4-二ohb以扭曲方向结合，使其中一个酚氧与天冬酰胺形成非常强的氢键，从而稳定酚酸并与黄素的π-系统产生电荷转移相互作用，从而产生绿色。
Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotate in the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in all structurally characterized DHODs, suggesting that the prospects for identifying a class-specific inhibitor directed against this site are poor. Nonetheless, two compounds that bind specifically to the Class 1A DHOD fromLactococcus lactis, 3,4-dihydroxybenzoate (3,4-diOHB) and 3,5-dihydroxybenzoate (3,5-diOHB), have been identified [Palfey et al. (2001)J. Med. Chem.44, 2861−2864]. The mechanism of inhibitor binding to the Class 1A DHOD fromL. lactishas now been studied in detail and is reported here. Titrations showed that 3,4-diOHB binds more tightly at higher pH, whereas the opposite is true for 3,5-diOHB. Isothermal titration calorimetry and absorbance spectroscopy showed that 3,4-diOHB ionizes to the phenolate upon binding to the enzyme, but 3,5-diOHB does not. The charge-transfer band that forms in the 3,4-diOHB complex allowed the kinetics of binding to be observed in stopped-flow experiments. Binding was slow enough to observe from pH 6 to pH 8 and was (minimally) a two-step process consisting of the rapid formation of a complex that isomerized to the final charge-transfer complex. Orotate and 3,5-diOHB bind too quickly to follow directly, but their dissociation kinetics were studied by competition and described adequately with a single step. Crystal structures of both inhibitor complexes were determined, showing that 3,5-diOHB binds in the same orientation as orotate. In contrast, 3,4-diOHB binds in a twisted orientation, enabling one of its phenolic oxygens to form a very strong hydrogen bond to an asparagine, thus stabilizing the phenolate and causing charge-transfer interactions with the π-system of the flavin, resulting in a green color.