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

1.时间分辨x射线晶体学结合了两种动力学晶体学技术，即衬底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。在反应开始之前，我们确定了酶-底物复合物的晶体结构：托品酮还原酶- ii - nadph -托品酮。复杂。本研究阐明的酶-底物配合物与我们之前研究的酶-产物配合物的结构比较表明，其中的一种底物，即托品酮，相对于产物是旋转的，从而使活性位点的空间组织有利于反应的进行。在原子分辨率下，研究了天然半乳糖醛酸和两种半乳糖醛酸配合物状态下的半乳糖醛酸内聚酶晶体结构。这些复合物的活性位点结构提供了对逆转糖基水解酶的机制的深入了解，也揭示了28家族与其他逆转糖基水解酶之间的差异。我们测定了烟草中含有钙调素结合域与Ca(2+)-钙调素络合物的玉米丙酮酸磷酸二激酶、含有钙调素结合域的MARCKS肽、含有Ca(2+)-钙调素络合物的肉豆醇化的CAP-23/ nap - 22n末端结构域以及光系统II的PsbP蛋白的晶体结构。

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.