【作者】 张敏；

【导师】 汤丽霞；

【作者基本信息】 电子科技大学 ， 生物学， 2022， 硕士

【摘要】 卤醇脱卤酶(HHDHs)可以高效催化邻卤醇分子内亲核取代脱卤反应形成相应的环氧化物,也可在亲核试剂作用下催化环氧化物开环反应。因此,该酶在制备光学纯β-取代醇等一系列手性药物中间体以及有机卤代污染物治理等方面都有着不可替代的作用。HheC晶体结构的解析为阐明其催化机理奠定了基础。HheC以同源四聚体形式存在,主要依靠催化三联体Ser132/Tyr145/Arg149行使催化功能。此外,在酶催化位点周围围绕着4条Loop,对酶分子的催化功能有着不同程度的影响:Loop1、Loop2上的氨基酸侧链与底物结合口袋氢键网络紧密相连;Loop3为卤离子结合位点,与酶催化过程的限速步关系密切;Loop4区域的氨基酸与底物释放通道的构成息息相关,并且作为HheC独有的一段氨基酸序列,对酶稳定性有显著影响。本篇论文在已有研究的基础上以4条Loop为研究中心,采用实验与计算生物学相结合手段,针对与催化反应相关的重要位点,采用迭代饱和突变策略将位于4条Loop上的重要氨基酸位点相互叠加构建12个ISM文库,以1,3-DCP为模式底物筛选近5300个样本,得到8个活性明显提升的突变体,其中活性提升最高的样品12-3(P84A-F86A-T134L-W139V)酶活高达野生型的8.4倍。通过对上述筛选结果分析表明,除Loop3外任意两环的叠加文库均得到优势突变体,以此推测Lo op1、Loop2、Loop4之间在酶催化作用中存在一定叠加作用。借助计算机手段构建突变体晶体结构、模拟分子对接、预测酶内部隧道走向等,分析突变体活性提升的原因及Loop之间在结构上的关联。宏观来看,突变位点的引入改变了Loop之间的氢键作用网络,氢键的缺失使得底物口袋明显柔性增大,疏水范围扩大,并且P84、F86、W139、W249位点均偏好小侧链氨基酸,位点叠加后酶底物口袋的空间位阻减小,有利于卤离子释放,使得酶催化能力明显增强。CAVER酶分子内部隧道预测结果显示,W139位氨基酸突变后,隧道tunnel-004不再在此区域转变方向,而是笔直的通向溶剂环境中,隧道出口转向结构疏松、柔性大的Loop4区域,整体走向平缓,更加有利于卤离子进出酶分子内部,从而使得催化效率提升,14-1、41-1中出现类似的现象,初步证明Loop1、Loop2、Loop4区域内氨基酸在HheC催化过程中起到一定的累积作用。更多还原

【Abstract】 Halohydrin dehalogenases(HHDHs)efficiently catalyze the dehalogenation of vicinal halo alcohols to epoxide by the intramolecular substitution mechanism.Also,HheC can catalyze epoxide ring-opening reaction with a series of anion nucleophiles.Therefore,the enzyme plays an irreplaceable role in the preparation of chiral pharmaceutical intermediates such as optically pure β-substituted alcohols.It also has applications in the degradation of polluting organic halides.The crystal structure of HheC lays the foundation for studying the catalytic mechanism.The homotetramer enzyme HheC performs catalytic function relying on the Ser132/Tyr145/Arg149 catalytic triad.In addition,four loops around the catalytic center of the enzyme have different degrees of influence on the catalytic function.Loop1 and loop2 regions are close to the hydrogen bond network of the substrate-binding pocket.The loop3 region is the halide ion binding area,which is closely related to the rate-limiting step of the enzyme catalysis process;the loop4 region,a unique amino acid sequence of HheC,is closely related to the composition of the substrate release channel.It has a significant effect on enzyme stability.According to existing research,four loops are selected as research points.This thesis combined with experimental method and computational biology selected 8 key sites,and constructed 12 ISM libraries.After screening nearly 5300 samples with1,3-DCP as a model substrate,8 brilliant variants were obtained,which the best one12-3(P84A-F86A-T134L-W139V)displaying approximately 8.4-fold higher activity than the wild-type enzyme.The results showed that the library formed by any two loops except loop3 yielded dominant mutation.Therefore,it is speculated that loop1,loop2,and loop4 have interaction during catalysis.To further explore the structural relationship between the loops,the thesis constructed the crystal structure of the mutation,simulated molecular docking,and predicted the internal tunnel of the enzyme via computer software.The analysis results showed that the amino acid changed the hydrogen bonding network between loops.Also,the absence of hydrogen bonds made the substrate pocket more flexible and the hydrophobic range expands,the P84,F86,W139,and W249 sites all prefer small side-chain amino acids.After site overlapping,the steric hindrance of the enzyme-substrate pocket is reduced,which is conducive to the release of halide ions.In addition,the catalytic ability of the enzyme is significantly enhanced.The predicted results of the CAVAR internal tunnel of the enzyme indicated that the mutated of 139 changes the orientation of tunnel-004.The tunnel no longer turns around at the site but goes straight into the solvent environment.Put the exit turns to the loop4 region simultaneously to obtain a new export with a loose structure and great flexibility.At the same time,the increased tunnel space is more conducive to the entry and exit of halide ions into the enzyme molecule,thereby improving the catalytic efficiency.Similar phenomena appeared in the structures of 14-1 and 41-1.These are sufficient to prove that the amino acids in the loop1,loop2,and loop4 regions interact in the catalytic process of HheC.更多还原