iso-Orotate decarboxylase (IDCase), which is involved in the thymidine salvage pathway, has attracted considerable interest owing to its chemical similarity to a hypothetical DNA decarboxylase in mammals. Although valuable insights into the active DNA demethylation of 5-methyl-cytosine can be obtained from the decarboxylation mechanism of 5-carboxyl-uracil (5caU) catalyzed by IDCase, this mechanism remains under debate. In this study, the catalytic mechanism of 5caU decarboxylation by IDCase was studied using hybrid quantum mechanics/molecular mechanics (QM/MM) methodologies and density functional theory (DFT) calculations with a truncated model. The calculations supported a mechanism involving three sequential stages: activation of the 5caU substrate via proton transfer from an arginine (R262′) to the carboxyl group of 5caU, formation of a tetrahedral intermediate, and decarboxylation of the tetrahedral intermediate to generate uracil as the product. The reaction pathways and structures obtained using the QM/MM and DFT methods coincided with each other. These simulations provided detailed insights into the unique mechanism of IDCase, clarifying various unresolved issues, such as the critical role of R262′. In addition, aspartate D323 was found to act as a general base in the tetrahedral intermediate formation step and a general acid in the later C–C bond cleavage step.

中文翻译：

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重新审视异乳清酸脱羧酶的脱羧机制


异乳清酸脱羧酶 (IDCase) 参与胸苷补救途径，由于其与哺乳动物中假设的 DNA 脱羧酶的化学相似性而引起了人们极大的兴趣。尽管可以从 IDCase 催化的 5-羧基-尿嘧啶 (5caU) 脱羧机制中获得对 5-甲基-胞嘧啶主动 DNA 去甲基化的有价值的见解，但该机制仍存在争议。在这项研究中，利用混合量子力学/分子力学（QM/MM）方法和截断模型的密度泛函理论（DFT）计算，研究了IDCase对5caU脱羧的催化机制。计算支持涉及三个连续阶段的机制：通过质子从精氨酸（R262'）转移到5caU的羧基来激活5caU底物，形成四面体中间体，以及四面体中间体脱羧以生成尿嘧啶作为产物。使用QM/MM和DFT方法获得的反应路径和结构彼此一致。这些模拟提供了对IDCase独特机制的详细见解，澄清了各种未解决的问题，例如R262'的关键作用。此外，还发现天冬氨酸 D323 在四面体中间体形成步骤中充当通用碱，并在随后的 C-C 键断裂步骤中充当通用酸。