一种新型环氧化物水解酶的发现及其催化机理的阐明

Discovery of a novel epoxide hydrolase and elucidation of its catalytic mechanism

摘要:

【目的】环氧化物水解酶(epoxide hydrolases,EHs)在手性药物的合成中起着重要作用。为了补充和发现更多的高性能环氧化物水解酶，通过基因调取技术探索新的环氧化物水解酶。【方法】通过基因调取技术鉴定了一种来自卡尔斯巴德曲霉的新型环氧化物水解酶(Aspergillus carlsbadensis epoxide hydrolase,AcEH)。采用AutoDock2预测AcEH的关键水解位点，通过计算设计阐明重要位点对AcEH的结构和催化机制的影响。【结果】对新型AcEH一级结构的分析揭示了3个特征性α/β EH基序的存在：HGWP、GYTFS和GGDIGS。AcEH酶表现出高活性，可在15 min内完全水解氧化苯乙烯(styrene oxide,SO)，比活性为13 951 U/g。K_m、V_max和k_cat/K_m值分别为(107.07±57.98) mmol/L、(37.22±17.85)μmol/(min·mg)和1.17 mmol/(L·s)。AcEH的关键水解位点是催化三联体的Asp192-His372-Glu346和2个保守酪氨酸Tyr251/314。某些突变(R49L、R49Y)导致酶失活，而其他突变(Y45L)导致无活性包涵体的形成。相互作用网络分析显示，第49个氨基酸残基的变化破坏了重要活性位点残基的相互作用，导致酶失活。另一方面，第45个氨基酸残基的改变使酶的结构不稳定，导致包涵体的形成。【结论】本研究发现了一种新的环氧化物水解酶，并分析了其水解机制，为进一步研究这种酶及其工业化应用提供了有价值的见解。

Abstract:

[Objective] Epoxide hydrolases (EHs) play a key role in the synthesis of chiral pharmaceuticals. We explored new EHs by engineering or gene retrieval, aiming to enrich and discover more high-performance EHs. [Methods] A novel epoxide hydrolase (Aspergillus carlsbadensisepoxide hydrolase, AcEH) from Aspergillus carlsbadensis was identified by gene retrieval technology. We then used AutoDock2 to predict the key hydrolysis sites of AcEH and employed computational design to clarify the influences of important sites on the structure and catalytic mechanism of AcEH. [Results] The primary structure of the novel EH had three characteristic α/β EH motifs: HGWP, GYTFS, and GGDIGS. AcEH exhibited high activity and could completely hydrolyze styrene oxide (SO) within 15 min, with a specific activity of 13 951 U/g. The K_m, V_max, and k_cat/K_m of AcEH were (107.07±57.98) mmol/L, (37.22±17.85) μmol/(min·mg), and 1.17 mmol/(L·s), respectively. The key hydrolysis sites of AcEH were Asp192-His372-Glu346, which catalyzed the triad, and two conserved tyrosine residues, Tyr251/314. The mutations R49L and R49Y caused enzyme inactivation, while the mutation Y45L resulted in the formation of inactive inclusion bodies. The interaction network revealed that changes in the 49th amino acid residue disrupted the interactions between key active site residues, leading to enzyme inactivation. On the other hand, the alteration of the 45th amino acid residue destabilized the enzyme structure, leading to the formation of inclusion bodies. [Conclusion] This study discovered a novel EH and analyzed its hydrolysis mechanism. The findings provide valuable insights for further research and engineering on this enzyme.