α-酮戊二酸依赖的加氧酶是在生物界广泛存在的最大酶家族之一，在生物合成与代谢、疾病的发生发展等方面具有重要作用。因此，该酶的性质改造和抑制剂筛选分别是生物制造和药物设计的重要内容。然而，由于目前常规的结构分析缺乏动态信息，阻碍了相关应用研究的发展。时间分辨晶体学可以在纳秒尺度捕捉化学反应的瞬时中间体并揭示反应过程的动态结构。但由于只有光激活的反应才能精确确定反应时间零点，所以目前只适用于极少数蛋白。本项目拟利用特有的非天然氨基酸标记技术，将普通生化反应改造为光激活反应，并将此技术应用于α-酮戊二酸依赖的酶家族成员。通过测定时间分辨晶体结构，捕捉底物到产物过程中的瞬时中间体及其相应的酶分子氨基酸修饰和构象变化过程，进一步阐明α-酮戊二酸依赖酶的催化机制。本项目研究不仅能拓展时间生物大分子时间分辨晶体学的样品适应性，也将为α-酮戊二酸依赖酶的工程改造和抑制剂筛选提供更精确的结构证据。

α-Ketoglutaric acid (α-KG) dependent oxygenases, which play important roles in biosynthesis, metabolism and pathogenesis, are one of the largest enzyme families widely existing in the life world. Therefore the enzyme engineering and inhibitor screening targeting to α-KG oxygenases are critical to bio-production and drug design. However, lack of the dynamic information in ordinary structure analysis hampered the related application studies. Time-resolved crystallography is a powerful technique to capture the intermediates in chemical reactions and depict the dynamic conformational changes in nanosecond time scale. However, the starting time point could be accurately determined only in photoactive reactions and few proteins are applicable. This project aims to make conversional enzymatic reaction to be photo-reactive by genetic incorporation of unnatural amino acid and apply this novel technique to α-KG oxygenases. The catalytic reaction process will be recorded by time-resolved crystallography and the mechanism of several typical α-KG oxygenases will be studied by capturing the short-lifetimed reactive intermediates and corresponding conformational changes. This project will not only expand the sample adaptability of protein time-resolved crystallography, but also provide direct evidence for α-KG oxygenase engineering and inhibitor design.