NAD(P)+-依赖型葡萄糖脱氢酶底物识别分子机制研究

项目名称： NAD(P)+-依赖型葡萄糖脱氢酶底物识别分子机制研究

项目编号： No.31200599

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 生物物理、生化与生物分子学、生物力学与组织工程

项目作者： 丁海涛

作者单位： 浙江大学

项目金额： 23万元

中文摘要： 葡萄糖脱氢酶底物谱广泛的特性限制了其在血糖即时监测方面的应用。但是对其底物特异性的改造至今仍受限于缺乏酶-底物复合物的结构信息。本项目以葡萄糖脱氢酶LsGDH为研究对象，结合蛋白质工程、蛋白质结晶和生物信息学方法，从静态和动态的角度研究酶和底物之间相互作用分子水平上的细节本质：(1)通过对无活性但保留底物结合能力的突变体进行结晶、衍射和解析，获得酶-辅酶-底物复合物的晶体结构信息；(2)通过分子动力学模拟，获取酶和底物在结合区及底物通道处的相互作用信息；(3)通过对可能影响底物特异性的氨基酸位点进行突变分析，明确起决定性作用的位点。(4)结合上述结果，阐明葡萄糖脱氢酶的底物识别机制。研究成果既有助于阐明葡萄糖脱氢酶底物识别过程中分子水平上的本质所在，也可以加深我们对于蛋白质和小分子相互作用机制细节的理解，拓展我们的研究思路。同时本项目研究结果也可为其他酶的底物特异性改造提供参考。

中文关键词： 葡萄糖脱氢酶；底物识别机制；结构；分子动力学；

英文摘要： The wide substrate spectrum of glucose dehydrogenase limits its application in real-time glucose measurement. However, changes in the substrate specificity of glucose dehydrogenase are still limited by the lack of structural information of enzyme-substrate complex. The project aims to use glucose dehydrogenase LsGDH as the research object to investigate the interactions between the enzyme and the substrates on the molecular level from static and dynamic angles by integrating protein engineering, protein crystallography and bioinformatics methods. The project can be divided into four parts as follows: (1) To obtain enzyme-coenzyme-substrate complex structure information through crystallization, X-ray diffraction and analysis of the nonfunctional mutant of LsGDH which retains the ability of binding substrate; (2) To obtain the interactions information at substrate binding site and substrate entrance channel of LsGDH by molecular dynamics simulations; (3) To clarify the sites significantly affecting the substrate specificity of glucose dehydrogenase by mutating the potential amino acid sites affecting the substrate specificity of glucose dehydrogenase; (4) To elaborate the substrate recognition mechanism of the glucose dehydrogenase based on above- mentioned results. The project can elaborate the essence of the su

英文关键词： glucose dehydrogenase；substrate recognition mechanism；structure；molecular dynamics；