Interaction of Benzoate Pyrimidine Analogues with Class 1A Dihydroorotate Dehydrogenase from Lactococcus lactis

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• 作者：Abigail E. Wolfe ; Majbritt Thymark ; Samuel G. Gattis ; Rebecca L. Fagan ; Yu-chen Hu ; Eva Johansson ; Susan Arent ; Sine Larsen ; Bruce A. Palfey
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：May 15, 2007
• 年：2007
• 卷：46
• 期：19
• 页码：5741 - 5753
• 全文大小：494K
• 年卷期：v.46,no.19(May 15, 2007)
• ISSN：1520-4995

文摘

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotatein the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in allstructurally characterized DHODs, suggesting that the prospects for identifying a class-specific inhibitordirected against this site are poor. Nonetheless, two compounds that bind specifically to the Class 1ADHOD from Lactococcus 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 ofinhibitor binding to the Class 1A DHOD from L. lactis has now been studied in detail and is reportedhere. Titrations showed that 3,4-diOHB binds more tightly at higher pH, whereas the opposite is true for3,5-diOHB. Isothermal titration calorimetry and absorbance spectroscopy showed that 3,4-diOHB ionizesto the phenolate upon binding to the enzyme, but 3,5-diOHB does not. The charge-transfer band thatforms in the 3,4-diOHB complex allowed the kinetics of binding to be observed in stopped-flowexperiments. Binding was slow enough to observe from pH 6 to pH 8 and was (minimally) a two-stepprocess 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 bycompetition and described adequately with a single step. Crystal structures of both inhibitor complexeswere 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 hydrogenbond to an asparagine, thus stabilizing the phenolate and causing charge-transfer interactions with the-system of the flavin, resulting in a green color.