STRUCTURE AND FUNCTION OF A FLAVOPROTEIN DEHYDROGENASE

这项建议旨在扩大酰基-C0A的结构研究 脱氢酶，这是一个酶家族，参与第一个 脂肪酸分解代谢中的氧化步骤和脂肪酸的分解代谢 一些氨基酸。还建议确定其结构 电子转移黄素蛋白(ETF)是植物体内的生理氧化剂。 酰基-CioA脱氢酶，将还原的等价物输送到主要 线粒体呼吸链通过ETF-泛醌氧化还原酶。胖子 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、怀孕和 分别为糖尿病。这些脱氢酶和ETF的关键作用 在新陈代谢中由人类疾病的严重程度来说明 这些脱氢酶和ETF中的每一个都存在遗传缺陷。 中链酰辅酶A脱氢酶(MCAD)的晶体结构 猪肝，有底物和无底物，以及两个短的结构 已经测定了链特异性酶。建议延长 对这三种酶的定点突变体和其他 脱氢酶家族的成员，包括长链酰基辅酶A 脱氢酶(LCAD)和异戊基辅酶A脱氢酶(IVD)。LCAD和IVD 是独一无二的，因为它们是甘氨酸残基，而不是在 已知序列的所有其他脱氢酶。高分辨率 获得了IVD和LCAD晶体的数据集。的结构 LCAD和IVD将确认/确定其催化残留物 分子建模和定点突变，还将揭示 这一独特替代的结构基础。两个版本的比较 这个脱氢酶家族的不同成员的结构将 使我们能够研究这些酶和 它们的酰基辅酶A底物及其分子基础 具体细节。并对MCAD的作用机理进行了探讨。 使用几种抑制剂，包括亚甲基环丙基-乙酰辅酶A， 一种低甘氨酸的代谢物，可导致牙买加人呕吐。 人的ETF已经被克隆、表达并以一种合适的形式结晶 用于高分辨率X射线分析。3.5A电子密度图 已取得解决方案，并正在进行进一步的阶段改进。 ETF及其各种酰基的高分辨结构 C0A，一种低甘氨酸的代谢物，会导致牙买加人呕吐。 人的ETF已经被克隆、表达并以一种合适的形式结晶 用于高分辨率X射线分析。3.5A电子密度图 已取得解决方案，并正在进行进一步的阶段改进。 ETF及其各种酰基的高分辨结构 辅酶A脱氢酶将使我们能够研究电子的分子基础 脱氢酶和ETF之间的转移，可能还有黄素蛋白- 黄素蛋白的相互作用。

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.