STRUCTURE AND FUNCTION OF A FLAVOPROTEIN DEHYDROGENASE

这一建议是为了扩展酰基-C 0A的结构研究 酶，一个酶家族，参与了第一个 在脂肪酸催化剂中的氧化步骤和在脂肪酸催化剂中的氧化步骤 一些氨基酸。 还建议确定 电子转移黄素蛋白（ETF），生理氧化剂， 酰基-CioA脱氢酶，其将还原当量汇集到主要的 线粒体呼吸链通过ETF-泛醌氧化还原酶。 脂肪 酸氧化是肝脏，心脏， 肾脏和骨骼肌。 这个过程的速度可以通过以下方式改变： 饮食，生理状态和疾病，如饥饿，怀孕， 糖尿病，分别。 这些资产和ETF的关键作用 在新陈代谢是说明了人类疾病的严重性归因 这些酶和ETF的遗传缺陷。 对中链酰基辅酶A脱氢酶（MCAD）的晶体结构进行了研究。 猪肝，有和没有基板，和两个短的结构， 链特异性酶已被确定。 建议延长 这些研究涉及这三种酶的定点突变体和其他 脱氢酶家族成员，包括长链酰基辅酶A 脱氢酶（LCAD）和异戊酰辅酶A脱氢酶（IVD）。 LCAD和IVD 它们的独特之处在于，它们含有甘氨酸残基，而不是谷氨酸， 已知序列的所有其他内切酶。 高分辨率 获得了IVD和ICAD晶体的数据集。 的结构 lCAD和iVD将确认/确定其通过以下方式识别的催化残留物： 分子建模和定点突变，也将揭示 这种独特替代的结构基础。 比较 该酶家族的不同成员的结构将 使我们能够研究这些酶之间的详细相互作用， 它们的酰基辅酶A底物及其底物的分子基础 特殊性 并对MCAD的机理进行了探讨 使用几种抑制剂，包括亚甲基环丙基-乙酰辅酶A， 一种引起牙买加呕吐病的次甘氨酸的代谢物。 人ETF已被克隆、表达并以合适的形式结晶， 用于高分辨率X射线分析。 3.5 A的电子密度图 已解决问题，并正在进一步改进阶段。 ETF的高分辨率结构与各种酰基的结构一起 C 0A，一种引起牙买加呕吐病的低甘氨酸代谢物。 人ETF已被克隆、表达并以合适的形式结晶， 用于高分辨率X射线分析。 3.5 A的电子密度图 已解决问题，并正在进一步改进阶段。 ETF的高分辨率结构与各种酰基的结构一起 CoA脱氢酶将使我们能够研究电子的分子基础 转移酶和ETF之间，也许，黄素蛋白， flavoprotein相互作用in general一般.

This proposal is to extend the structural studies of acyl-C0A dehydrogenases, a family of enzymes that are involved in both the first oxidative step in the catabolism of fatty acids and in the catabolism of some amino acids. It is also proposed to determine the structure of electron transfer flavoprotein (ETF), the physiological oxidant of the acyl-CioA dehydrogenases, that funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase. Fatty acid oxidation is the principal energy yielding process in liver, heart, kidney, and skeletal muscle. The rate of this process can be altered by diet, physiological status, and disease, such as starvation, pregnancy, and diabetes, respectively. The critical roles of these dehydrogenases and ETF in metabolism is illustrated by the severity of human diseases attributive to inherited deficiencies of each of these dehydrogenases and ETF. The crystal structures of medium chain acyl CoA dehydrogenase (MCAD) from pig liver, with and without substrates, and the structures of two short chain-specific enzymes have been determined. It is proposed to extend these studies to site-directed mutants of these three enzymes and to other members of the dehydrogenase family, including long chain acyl-CoA dehydrogenase (LCAD) and isovaleryl-CoA dehydrogenase (IVD). LCAD and IVD are unique in that they a glycine residue in lieu of the glutamate found in all other dehydrogenases for which sequences are known. A high resolution data set of IVD and crystals of lCAD have been obtained. The structures of lCAD and iVD will confirm/determine their catalytic residues identified by molecular modeling and site-directed mutagenesis and also will reveal the structural basis for this unique substitution. Comparison of the structures of different members of this family of dehydrogenases will enable us to study the detailed interactions between these enzymes and their acyl-CoA substrates and the molecular basis of their substrate specificities. It is also proposed to probe the mechanism of the MCAD action using several inhibitors, including methylenecyclopropyl-acetyl-CoA, a metabolite of hypoglycine that causes Jamaican vomiting sickness. Human ETF has been cloned, expressed, and crystallized in a form suitable for high resolution X-ray analysis. An electron density map at 3.5 A resolution has been obtained and further phase refinements are in progress. The high resolution structure of ETF together with those of various acyl C0A, a metabolite of hypoglycine that causes Jamaican vomiting sickness. Human ETF has been cloned, expressed, and crystallized in a form suitable for high resolution X-ray analysis. An electron density map at 3.5 A resolution has been obtained and further phase refinements are in progress. The high resolution structure of ETF together with those of various acyl CoA dehydrogenases will enable us to study the molecular basis of electron transfer between the dehydrogenases and ETF and, perhaps, flavoprotein- flavoprotein interactions in general.