卤醇脱卤酶催化环氧化物立体选择性开环反应规律的研究

Halohydrin dehalogenases may become attractive catalysts for the facile preparation of enantiopure β-hydroxy nitriles from racemic epoxides, which is an important way to replace and expand traditional organic synthesis for their significant advantages as high selectivity, moderate reaction condition and environmental benign. Due to the lack of deep understanding of substrate specificity and stereoselectivity law, the development, application and reconstruction of halohydrin dehalogenase are severely restricted. This project focused on the asymmetric ring opening of epoxides. By establishing typical halohydrin dehalogenase library, structure library and varied substituent group library, together with enzyme activity fingerprint analysis system with high resolution, the fingerprint map of substrate specificity and stereoselectivity can be obtained. The homologous modeling, molecular docking technologies and site-directed mutagenesis will be further applied to study the effects of key amino acid sites of halohydrin dehalogenase structure on the activity and stereoselectivity to different substrates, to accurately position the amino acid residues and functional domains that affects the halohydrin dehalogenase, substrate recognition, stereoselective recognition, which can finally reveal the molecular mechanisms of its substrate specificity and stereoselectivity. The whole research provide a better understanding of stereoselective ring opening of epoxides catalyzed by halohydrin dehalogenases and promote their application in pharmaceutical synthesis.

卤醇脱卤酶催化环氧化物不对称开环制备手性β-取代醇具有高选择性、条件温和、环境友好等优点，成为可持续发展过程中替代或拓展有机化学合成的重要方法。由于对其不对称开环反应规律认识的匮乏，卤醇脱卤酶在不对称催化合成中的开发应用受到限制。本项目通过构建典型的卤醇脱卤酶酶库和结构多样的环氧化物底物文库，建立高分辨率卤醇脱卤酶酶活力指纹分析体系，获得卤醇脱卤酶底物特异性和立体选择性指纹图谱。基于酶活指纹图谱信息，综合运用同源建模、分子对接及定点或饱和突变技术，研究影响卤醇脱卤酶催化活性和立体选择性的关键位点，进一步定位影响卤醇脱卤酶底物和立体选择性识别的氨基酸残基和结构功能域，揭示卤醇脱卤酶底物特异性和立体选择性规律。本项目研究不仅有助于丰富对卤醇脱卤酶催化不对称开环的认识，同时对拓展卤醇脱卤酶在有机化学领域的应用、促进生物催化合成手性β-取代醇的发展将产生一定的推动作用。

立体选择性卤醇脱卤酶是一种重要的手性合成工具酶,在制备手性环氧及手性β-取代醇方面具有广阔的应用前景,日益受到重视。但是由于对卤醇脱卤酶催化立体选择性开环反应分子机制认识的匮乏，严重制约了其在手性中间体合成中的开发应用。课题利用卤醇脱卤酶的高度保守序列，基于数据库挖掘法构建了小型的卤醇脱卤酶酶库，经功能筛选获得9个新型卤醇脱卤酶。选择具有重要应用前景的4个卤醇脱卤酶，考察了新型卤醇脱卤酶催化缩水甘油醚类环氧底物开环反应的底物特异性、催化动力学和立体选择性。以苯基缩水甘油醚为模式底物，借助于同源建模、分子对接、结构及同源序列比对定位了影响卤醇脱卤酶立体选择性的关键位点，通过单点突变和组合突变获得了多个催化选择性提高或者逆转的高效突变体。以结构、取代基多样的环氧化合物为底物，通过研究引起立体选择性改变的酶分子结构基础，揭示了立体选择性卤醇脱卤酶不同突变点位置对不同取代基团，不同取代位置环氧底物立体选择性的影响规律，准确了定位影响卤醇脱卤酶底物识别、立体选择性识别的氨基酸残基。项目研究成果不仅为卤醇脱卤酶理性设计奠定理论基础，也进一步拓宽立体选择性卤醇脱卤酶的应用范围。