Forefront X-Ray Crystallography of Enzymes by using the third generation synchrotron radiation

1.时间分辨的X射线晶体学两种动力学晶体学技术的结合，即底物的连续流动和劳厄衍射测量，使我们能够在反应进行之前捕获过渡结构。我们在反应开始前测定了酶-底物复合物的晶体结构：托品酮还原酶-Ⅱ-NADPH-托品酮。复杂.本研究中阐明的酶-底物复合物与我们先前研究中的酶-产物复合物的结构比较表明，底物之一托品酮，相对于产物旋转，使活性位的空间结构有利于反应的进行。X射线晶体学在原子分辨率下解决了来自Stereum purpureum的内切多聚半乳糖醛酸酶在天然和两种半乳糖醛酸络合物状态下的晶体结构。复合物的活性中心结构为糖基水解酶的转化机制提供了新的思路，同时也揭示了28家族糖基水解酶与其他糖基水解酶的差异。3. X射线晶体结构测定与分析困难我们测定了玉米丙酮酸磷酸二激酶的晶体结构，含有与Ca（2+）-钙调蛋白复合的钙调蛋白结合结构域的MARCKS肽、与Ca（2+）钙调蛋白复合的肉豆蔻酰化CAP-23/NAP-22 N-末端结构域和来自烟草的光系统II的PsbP蛋白。

1.time-resolved x-ray crystallography A combination of two kinetic crystallographic techniques, a continuous flow of the substrates and Laue diffraction measurement, enabled us to capture the transit structures prior to the reaction proceeding. We determined the crystal stuructre of enzyme-substrate complex prior to reaction initiation: tropinone reductase-II-NADPH-tropinone. complex. A structural comparison of the enzyme-substrate complex elucidated in this study with the enzyme-product complex in our previous study indicated that one of the substrates, tropinone, is rotated relative to the product so as to make the spatial organization in the active site favorable for the reaction to proceed.2.ultra-high-resolution X-ray crystallography Crystal structures of endopolygalacturonase from Stereum purpureum were solved in native and two galacturonic acid complex states at atomic resolution. The active-site architecture of the complexes provides insight into the mechanism of inverting glycosyl hydrolases and also sheds light on the basis of the differences between the family 28 and the other inverting glycosyl hydrolases.3.X-ray crystal structure determination with difficult analysis We determined crystal structure of maize pyruvate phosphate dikinase, a MARCKS peptide containing the calmodulin-binding domain in complex with Ca(2+)-calmodulin, a myristoylated CAP-23/NAP-22N-terminal domain complexed with Ca(2+)calmodulin, and the PsbP protein of photosystem II from Nicotiana tabacum.