一种新型环氧化物水解酶的发现及其催化机理的阐明(英文)  

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

作　　者：朱美南 谷笑 柳得楷 张凌志 赵淑燕 张礼娟 张光亚[1] 江伟 ZHU Meinan;GU Xiao;LIU Dekai;ZHANG Lingzhi;ZHAO Shuyan;ZHANG Lijuan;ZHANG Guangya;JIANG Wei(College of Chemical Engineering,Huaqiao University,Xiamen 361021,Fujian,China;State Key Laboratory of Microbial Technology,Shandong University,Qingdao 266237,Shandong,China)

机构地区：[1]华侨大学化工学院,福建厦门361021 [2]山东大学,微生物技术国家重点实验室,山东青岛266237

出　　处：《微生物学报》2025年第1期389-401,共13页Acta Microbiologica Sinica

基　　金：国家自然科学基金(21808073);福建省自然科学基金(2021J05058);微生物技术开放项目国家重点实验室基金(M2022-02);中央高校基本科研业务费专项资金(ZQN-814);华侨大学高层次人才科研启动费(600005-Z17Y0072);泉州市科技计划(2018C008)。

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,Ac EH)。采用Auto Dock2预测Ac EH的关键水解位点,通过计算设计阐明重要位点对Ac EH的结构和催化机制的影响。【结果】对新型Ac EH一级结构的分析揭示了3个特征性α/βEH基序的存在:HGWP、GYTFS和GGDIGS。Ac EH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13951 U/g。Km、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)。Ac EH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。[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 carlsbadensis epoxide 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 characteristicα/βEH motifs:HGWP,GYTFS,and GGDIGS.AcEH exhibited high activity and could completely hydrolyze styrene oxide(SO)within 15 min,with a specific activity of 13951 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.

关 键 词：生物催化 环氧化物水解酶 手性药物 分子动力学模拟 分子对接 水解机理 

分 类 号：Q55[生物学—生物化学]