The catalytic mechanism of limonene epoxide hydrolase (LEH) was investigated theoretically using the density functional theory method B3LYP. LEH is part of a novel limonene degradation pathway found in Rhodococcus erythropolis DCL14, where it catalyzes the hydrolysis of limonene-1,2-epoxide to give limonene-1,2-diol. The recent crystal structure of LEH was used to build a model of the LEH active site composed of five amino acids and a crystallographically observed water molecule. With this model, hydrolysis of different substrates was investigated. It is concluded that LEH employs a concerted general acid/general base-catalyzed reaction mechanism involving protonation of the substrate by Asp101, nucleophilic attack by water on the epoxide, and abstraction of a proton from water by Asp132. Furthermore, we provide an explanation for the experimentally observed regioselective hydrolysis of the four stereoisomers of limonene-1,2-epoxide.

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柠檬烯环氧水解酶催化机理的理论研究

使用密度泛函理论方法 B3LYP 从理论上研究了柠檬烯环氧化物水解酶 (LEH) 的催化机理。LEH 是在红球菌 erythropolis DCL14 中发现的一种新的柠檬烯降解途径的一部分，它催化柠檬烯-1,2-环氧化物水解得到柠檬烯-1,2-二醇。LEH 的最新晶体结构用于构建由五个氨基酸和晶体学观察到的水分子组成的 LEH 活性位点模型。使用该模型，研究了不同底物的水解。得出的结论是，LEH 采用协调的通用酸/通用碱催化反应机制，包括 Asp101 对底物的质子化、水对环氧化物的亲核攻击以及 Asp132 从水中提取质子。此外，