Structure and Function of a Flavoprotein Dehydorgenase

描述(由申请人提供)： 这一建议扩展了酰基辅酶A脱氢酶电子转移黄素蛋白(ETF)和ETF-泛醌氧化还原酶(ETF-QO)的结构研究。ADS是一个酶家族，参与线粒体脂肪酸的第一步氧化和某些氨基酸的分解代谢。ETF是ADS和肌氨酸脱氢酶的生理氧化剂。肌氨酸脱氢酶不是AD家族的成员，在胆碱代谢中发挥作用，它将一个1碳单位转移到四氢叶酸。ETF通过ETF-QO，一种膜结合的铁硫黄素蛋白，将还原等价物输送到主要的线粒体呼吸链。脂肪酸氧化是肝脏、心脏和肌肉的主要能量产生过程。这一过程的速度可以通过饮食(饥饿)、生理状态(怀孕)或疾病(糖尿病)来改变。ADS、ETF、ETF-QO和肌氨酸脱氢酶的关键代谢作用可以从这些酶遗传缺陷引起的人类疾病的严重性来说明。对短链和中链酰辅酶A脱氢酶(MCAD)、异戊酰辅酶A脱氢酶和戊二酰辅酶A脱氢酶(GCD)的晶体结构进行了测定。建议：1)继续研究MCAD及其与底物类似物/抑制剂的复合物的突变体，确定人长链酰辅酶A脱氢酶的结构，以确定催化碱基，确定长链专一性的结构基础；2)继续GCD的结构研究，以研究脱羧基反应的机理；3)完成异丁酰辅酶A脱氢酶(参与缬氨酸代谢)和短/支链酰辅酶A脱氢酶(参与异亮氨酸分解)的结构测定，以确定它们的支链底物特异性。猪ETF-QO的结构已在结合和不结合泛醌的情况下确定。还建议：4)将ETF-QO的研究扩展到其与泛醌类似物和突变体的络合物中，以确定4Fe-4S簇的作用；5)启动肌氨酸脱氢酶的结构测定，以确定氧化脱甲基化和1-碳转移反应的机理。此外，还建议将不饱和脂肪酸代谢的辅助酶--大肠杆菌二烯酰辅酶A还原酶的结构研究扩展到其突变体和具有不同立体异构体底物的复合体，以确认可能的催化残基的作用，并确定不同立体异构体代谢的结构基础。

DESCRIPTION (provided by applicant): This proposal extends the structural studies of acyl-CoA dehydrogenases electron transfer flavoprotein (ETF), and ETF-ubiquinone oxidoreductase (ETF-QO). ADs are an enzyme family involved in the first step in mitochondrial fatty acid (-oxidation and in the catabolism of some amino acids. ETF is the physiological oxidant of ADs and sarcosine dehydrogenase. Sarcosine dehydrogenase is not a member of the AD family and functions in choline metabolism and transfers a 1-carbon unit to tetrahydrofolate. ETF funnels the reducing equivalents to the main mitochondrial respiratory chain via ETF- QO, a membrane-bound, iron-sulfur flavoprotein. Fatty acid (-oxidation is the major energy-producing process in the liver, heart, and muscle. The rate of this process can be altered by diet (starvation), physiological status (pregnancy), or diseases (diabetes). The critical metabolic roles of ADs, ETF, ETF-QO, and sarcosine dehydrogenase are illustrated by the severity of human diseases resulting from inherited deficiencies of these enzymes. Crystal structures have been determined for short- and medium-chain acyl-CoA dehydrogenases (MCAD), isovaleryl-CoA dehydrogenase, and glutaryl-CoA dehydrogenase (GCD). It is proposed: 1) to continue the studies of mutants of MCAD and its complexes with substrate analog/inhibitors and 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; 2) to continue the structural studies of GCD in order to study the mechanism of the decarboxylation reaction; and 3) to complete the structure determination of isobutyryl-CoA dehydrogenase (involved in valine metabolism) and short/branched chain acyl-CoA dehydrogenase (involved in isoleucine catabolism) to determine their branched chain-substrate specificities. Structure of porcine ETF-QO has been determined with and without a bound ubiquinone. It is also proposed: 4) to extend studies of ETF-QO to its complexes with ubiquinone analogs and mutants, in order to determine the role of the 4Fe-4S cluster and 5) to initiate the structure determination of sarcosine dehydrogenase in order to determine the mechanism of oxidative demethylation and 1-carbon transfer reactions. It is further proposed 6) to extend the structural studies of E. coli dienoyl-CoA reductase, an auxiliary enzyme for unsaturated fatty acid metabolism, to its mutants and complexes with different stereoisomeric substrates to confirm the roles of putative catalytic residues and to determine the structural basis for metabolism of different stereoisomers.