Demonstration School, University of Phayao, Phayao 56000, Thailand.
Unit of Excellence in Computational Molecular Science and Catalysis, and Division of Chemistry, School of Science, University of Phayao, Phayao 56000, Thailand.
Phys Chem Chem Phys. 2023 Mar 22;25(12):8767-8778. doi: 10.1039/d2cp05262h.
Dihydropyrimidinase (DHPase) is a key enzyme in the pyrimidine pathway, the catabolic route for synthesis of β-amino acids. It catalyses the reversible conversion of 5,6-dihydrouracil (DHU) or 5,6-dihydrothymine (DHT) to the corresponding -carbamoyl-β-amino acids. This enzyme has the potential to be used as a tool in the production of β-amino acids. Here, the reaction mechanism and origin of stereospecificity of DHPases from and CECT4114 were investigated and compared using a quantum mechanical cluster approach based on density functional theory. Two models of the enzyme active site were designed from the X-ray crystal structure of the native enzyme: a small cluster to characterize the mechanism and the stationary points and a large model to probe the stereospecificity and the role of stereo-gate-loop (SGL) residues. It is shown that a hydroxide ion first performs a nucleophilic attack on the substrate, followed by the abstraction of a proton by Asp358, which occurs concertedly with protonation of the ring nitrogen by the same residue. For the DHT substrate, the enzyme displays a preference for the L-configuration, in good agreement with experimental observation. Comparison of the reaction energetics of the two models reveals the importance of SGL residues in the stereospecificity of catalysis. The role of the conserved Tyr172 residue in transition-state stabilization is confirmed as the Tyr172Phe mutation increases the activation barrier of the reaction by ∼8 kcal mol. A detailed understanding of the catalytic mechanism of the enzyme could offer insight for engineering in order to enhance its activity and substrate scope.
二氢嘧啶酶 (DHPase) 是嘧啶途径中的关键酶，嘧啶途径是β-氨基酸合成的分解途径。它催化 5,6-二氢尿嘧啶 (DHU) 或 5,6-二氢胸腺嘧啶 (DHT) 可逆转化为相应的β-氨基甲酰-β-氨基酸。该酶有可能被用作生产β-氨基酸的工具。在这里，使用基于密度泛函理论的量子力学簇方法研究并比较了来自 和 CECT4114 的 DHPases 的反应机制和立体特异性起源。从天然酶的 X 射线晶体结构设计了两种酶活性位点模型：一个小簇用于描述机制和稳定点，一个大模型用于探测立体特异性和立体门控环 (SGL) 残基的作用。结果表明，首先是一个氢氧根离子对底物进行亲核攻击，然后由 Asp358 提取质子，这与同一残基对环氮的质子化同时发生。对于 DHT 底物，酶显示出对 L-构型的偏好，这与实验观察结果非常吻合。对两种模型的反应能学比较揭示了 SGL 残基在催化立体特异性中的重要性。保守的 Tyr172 残基在过渡态稳定中的作用得到了证实，因为 Tyr172Phe 突变使反应的活化能垒增加了约 8 kcal mol。对酶催化机制的深入了解可以为工程设计提供启示，以提高其活性和底物范围。
