发展基因密码子扩展及时间分辨X-射线晶体衍射方法研究氧化酶催化机理

Time resolved X-ray diffraction（TXRD）technique was used to determine the dynamic structure of the enzyme for studying of the catalytic mechanism. Oxidase is the key step for biological energy conversion and signal transduction. NADH oxidase (NOX) can reduce oxygen to superoxide anion, which is very important for the regulation of cell growth and immunity. Heme copper oxidase(HCO) can reduce oxygen to water, which is essential for bio-energy production. The bottleneck which limit the mechanistic investigation and application of these important oxidases are their large molecular weight, difficulty in expression, and difficulty for performing spectroscopic studies. We will use small and easily producible model protein as scaffold, install the catalytic center of NOX and HCO active sites into these model proteins, with the aim to mimic the function of complex oxidases with time-resolved crystal structure diffraction technique..Using the genetic code expansion method and the model enzyme approach, we will investigate the catalytic mechanism through which HCO and NOX reduce oxygen into water and superoxide, respectively. Relying on synchronous optical facilities, we will analysis of the structure of series of oxidase, the regulatory catalytic mechanism of metal centers’ coordination, valence and spin states. Such study will be helpful for rational designing of high efficient model oxidases and will be applicable in synthetic biology.To provide a high quality diffraction sample for synchrotron light source, it will also promote the research and development of the instrument with independent intellectual property rights.

时间分辨X-射线晶体衍射技术能够测定酶的动态结构，用于研究催化机理。氧化酶对生物体中的物质能量转化、信号转导起着关键的作用。NADH oxidase (NOX)将氧气还原为超氧阴离子，在调控细胞生长及免疫等过程中起重要作用； Heme copper oxidase(HCO) 还原氧气为水，是生物能量工厂的主要元件。目前制约这些酶机理研究和应用的主要瓶颈是它们分子量大，难表达。前期工作中，我们已实现了在小蛋白质特定位点引入非天然氨基酸用于模拟复杂氧化酶。.利用时间分辨晶体衍射技术研究氧化酶活性中心环境及催化机理，解析NOX及HCO具有不同活性的机制，是本研究的主要问题。依托同步光设施，我们将解析系列氧化酶的结构，研究金属中心的配位、价态、自旋态对催化活性的调控机制，实现高效氧化酶的理性设计及其在合成生物学领域的应用。同时，为同步光源提供高质量的衍射样品，将促进具有自主知识产权仪器的研发。

时间分辨X-射线晶体衍射技术能够测定酶的动态结构，用于研究催化机理。氧化酶对生物体中的物质能量转化、信号转导起着关键的作用。NADH oxidase (NOX)将氧气还原为超氧阴离子，在调控细胞生长及免疫等过程中起重要作用； Heme copper oxidase(HCO) 还原氧气为水，是生物能量工厂的主要元件。目前制约这些酶机理研究和应用的主要瓶颈是它们分子量大，难表达。前期工作中，我们已实现了在小蛋白质特定位点引入非天然氨基酸用于模拟复杂氧化酶。.依托同步光设施，本项目利用时间分辨晶体衍射技术研究了人工设计活性酶活性中心环境及催化机理，解析了具有不同活性的机制，实现了高效人工酶的理性设计及其在合成生物学领域的应用。同时，为同步光源提供高质量的衍射样品，也促进了具有自主知识产权仪器的研发。