维生素K2合成关键酶的结构解析与基于结构的抑制剂设计

With the increasingly urgent problem of bacterial drug resistance, search novel antibiotic target and develop corresponding inhibitor are the hot issues in drug development field. The menaquinone biosynthetic pathway is essential for bacteria survival. However, it is not available in human being. Thus, this pathway can be regarded as a target for antibiotics. This project set the key enzyme MenE in menaquinone biosynthetic pathway of Mycobacterium tuberculosis and Staphylococcus aureus as a research object. Combing previous enzyme over-expression and purification study, we plan to obtain both the “OPEN” and “CLOSE” form crystal structures of MenE by means of protein crystallization, synchrotron radiation, structure determination and refinement. Based on the solved structure, we will use computer assistant screening method such as database search, molecular docking, free Gibbs energy calculation to screen inhibitors which can inhibit each or both “OPEN” and “CLOSE” forms of MenE. Moreover, the substrate competitive inhibition experiment will be established to investigate the actual inhibition effects targeting MenE and pathogenic bacteria. Finally , we will introduce molecular dynamic simulation to disclose the inhibition mechanism. This work will lay the theoretical foundation for novel antibiotic development.

随着细菌耐药性问题的日益突出，寻找新的药物作用靶标并进行抑制剂设计是药物研发领域关注的热点。维生素K2合成途径是细菌存活所必需的，而人体内并不存在该途径，因此可以作为抗生素研发的靶标。本课题以结核分枝杆菌、金黄色葡萄球菌等重要病原微生物中维生素K2合成途径的关键酶MenE作为研究对象，结合前期的MenE高效表达和酶纯化基础，拟通过蛋白质晶体筛选与优化培养、同步辐射衍射、晶体结构解析与精修等方法首次获得MenE“OPEN”和“CLOSE”两种构象的高分辨率三维结构。基于结构进行以数据库搜索、分子对接、吉布斯自由能计算为手段的计算机辅助筛选，获得分别或同时作用于这两种构象的小分子酶抑制剂，进一步设计底物竞争性抑制实验，考察抑制剂对MenE活性以及病原菌生长的实际抑制效果，利用分子动力学模拟进一步揭示抑制机理，为该类新型抗生素的研发奠定理论基础。

维生素K2合成途径是细菌存活所必需的，而人体内并不存在该途径，因此可以作为新型抗生素研发的靶标，本课题以此为思路研究了枯草芽孢杆菌Bacillus subtilis合成途径中关键酶BsMenE的晶体结构。在本研究中，BsMenE的母体结构（apo form）被解析出来，分辨率1.98埃；BsMenE与ATP、AMP、OSB-AMP等小分子的复合物晶体结构也被解析出来，分辨率为2.60~2.82埃。通过分析这些结构，找到了影响BsMenE催化反应的关键残基，包括T152, G154, T155, T156, G157, H196, R382等，对这些残基进行了定点突变研究，掌握了它们对催化过程的影响规律。以上成果符合预期要求，所获得的实验数据和发表的论文专利将为本领域的深入研究提供技术借鉴。