STRUCTURE /FUNCTION OF A FLAVOPROTEIN DEHYDROGENASE

本研究旨在扩展酰基辅酶a脱氢酶及其生理电子转移伙伴电子转移黄蛋白（ETF）的结构研究。酰基辅酶a脱氢酶是一个酶家族，参与脂肪酸的线粒体分解代谢和一些氨基酸的分解代谢的第一个氧化步骤。ETF通过ETF-泛醌氧化还原酶（ETF- qo）（一种膜结合的铁硫黄蛋白）将还原等价物输送到主要的线粒体呼吸链；并提出了确定ETF-QO结构的方法。脂肪酸氧化是肝脏、肾脏和骨骼肌的主要能量生成过程。这一过程的速度可因饮食、生理状况和疾病而改变，例如饥饿、怀孕和糖尿病。酰基辅酶a脱氢酶ETF和ETF- qo在代谢中所起的关键作用，可以通过这些酶的遗传缺陷导致的人类疾病的严重程度来说明。已经确定了短链和中链酰基辅酶a脱氢酶及其突变体以及异戊酰和戊二酰辅酶a脱氢酶（GCD）的晶体结构。建议：继续研究MCAD突变体及其与底物类似物/抑制剂的复合物；测定人长链酰基辅酶a脱氢酶的结构，以确定催化碱基，确定长链特异性的结构基础；完成GCD结构的细化，研究脱羧反应的机理。GCD催化脱氢和脱羧反应。已经确定了人类ETF和细菌ETF的晶体结构。我们建议将这些研究扩展到突变型etf，以研究其与电子转移伙伴、脱氢酶和ETF-QO的相互作用。用x射线衍射法对Pig ETF-QO进行了结晶，并对其结构进行了测定。高分辨率的结构，以及ETF和脱氢酶的结构，将使我们能够研究这些黄蛋白之间电子转移途径的分子基础。

This proposal is to extend the structural studies of the acyl-CoA dehydrogenases and their physiological electron transfer partner, electron transfer flavoprotein (ETF). Acyl-CoA dehydrogenases are a family of enzymes that are involved in the first oxidative step in the mitochondrial catabolism of fatty acids and in the catabolism of some amino acids. ETF funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (ETF-QO), a membrane-bound, iron-sulfur flavoprotein; it is also proposed to determine the structure of ETF-QO. Fatty acid oxidation is the principal energy-yielding process in the liver, kidney, and skeletal muscle. The rate of this process can be altered by diet, physiological status, and disease, exemplified by starvation, pregnancy, and diabetes. The critical roles played in metabolism by acyl-CoA dehydrogenases, ETF, and ETF-QO, is illustrated by the severity of human diseases resulting from inherited deficiencies of each of these enzymes. Crystal structures have been determined for short- and medium-chain acyl-CoA dehydrogenases and their mutants and isovaleryl- and glutaryl-CoA dehydrogenase (GCD). It is proposed: to continue the studies of mutants of MCAD and its complexes with substrate analog/inhibitors; to determine the structure of human long chain acyl-CoA dehydrogenase in order to confirm the catalytic base and determine the structural basis for long-chain specificity; and to complete the refinement of the structure of GCD in order to study the mechanism of the decarboxylation reaction. GCD catalyzes both the dehydrogenation and decarboxylation reactions. Crystal structures of human ETF and a bacterial ETF have been determined. It is proposed to extend these studies to mutant ETFs to study the interactions with their electron transfer partners, the dehydrogenases and ETF-QO. Pig ETF-QO has been crystallized and its structure determination is in progress, using X-ray diffraction methods. The high resolution structure, together with those of ETF and the dehydrogenases, will enable us to study the molecular basis of electron transfer pathway between these flavoproteins.