X-ray structure analyses of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum and possible complexes with substrate, product as well as substrate and product analogues

本拨款申请的目的是测定氨基丁酸梭菌4-羟基丁基辅酶a脱水酶的x射线晶体结构。由于4-羟基丁基辅酶a脱水酶含有铁/硫簇，因此可以应用多波长异常色散（MAD）技术，为直接的x射线结构分析奠定基础。酶的结晶将在厌氧条件下进行。这种黄素-铁/硫酶的三维原子结构的知识将为理解这种特殊酶的机制作用，更好地解释光谱和动力学数据以及规划新的先进实验（例如，定点诱变，底物类似物/酶或抑制剂/酶复合物的晶体结构）提供合理的基础。关键的兴趣点是澄清是否真的在催化过程中选择性自由基中间体产生的酶。此外，结构测定将证明Fe-S簇是否彼此接近，并与FAD基团接近，从而允许Fe-S簇的光谱耦合以及Fe-S簇与FAS基团之间的相互作用。

The goal of this grant application is the determination of the X-ray crystal structure of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. As 4-hydroxybutyryl-CoA dehydratase contains iron/sulfur clusters the multiple wavelength anomalous dispersion (MAD) technique can be applied which forms the basis for a straightforward X-ray structure analysis. The crystallization of the enzyme will be performed under anaerobic conditions. The knowledge if the three-dimensional atomic structure of this flavin-iron/sulfur enzyme will form the rational basis for understanding the mechanistic action of this peculiar enzyme, a better interpretation of the spectroscopic and kinetic data and the planning of new advancing experiments (e.g. site-directed mutagenesis, crystal structures of substrate analogue/enzyme or inhibitor/enzyme complexes). The key point of interest is the clarification if really during catalysis selective radical intermediates are generated in the enzyme. Furthermore, the structure determination will prove if the Fe-S clusters are close to each other and to the FAD group allowing the spectroscopic coupling of the Fe-S clusters and the interaction between the Fe-S clusters and the FAS group.