Structure and Function of Enzymes in Fatty Acid Oxidation

描述（由申请人提供）：脂肪酸β-氧化是肝脏、心脏和肌肉中的主要能量产生过程。它是通过一系列的四个反应进行的，这些反应连续地将乙酰辅酶A从脂肪酰基辅酶A中裂解出来。这一过程的速度可以通过饮食（进食/禁食），生理状态（怀孕）或疾病（糖尿病）来改变。第一四个 该过程中的反应由黄素蛋白家族酰基辅酶A脱氢酶（AD）启动。电子转移黄素蛋白（ETF）和膜结合的ETF-泛醌氧化还原酶（ETF-QO）催化从AD到线粒体OXPHOS链的电子转移。存在至少七种用于催化短链酰基辅酶A的可溶性AD，以及两种特异于长链脂肪酰基辅酶A的膜结合AD，极长链AD（VLCAD）和ACAD 9。剩下的三个长链脂肪酸的β-氧化反应由三功能蛋白（TFP）进行，TFP是一种膜结合的多酶复合物。脂肪酸氧化的先天性缺陷已成为一个日益严重的健康问题，现在是通过扩大新生儿筛查确定的最常见的一组疾病，影响到全国每1000名出生婴儿中的2-3名。这些疾病表现为婴儿猝死综合症，引起心肌病，并且是年龄较大的儿童和年轻人骨骼肌病的最常见原因。最近，ACAD 9已被证明是组装复合物I所必需的，复合物I是五种OXPHOS复合物中最大和最复杂的酶（约980 kDa，具有45个亚基）。关于这种重要酶的组装过程的机制知之甚少。线粒体OXPHOS系统障碍是最常见的先天性代谢疾病，导致从运动不耐受到发育不良的各种临床表型。我们已经确定了除一种可溶性AD之外的所有可溶性AD以及一种膜结合AD（VLCAD）、ETF和ETF-QO的晶体结构。建议的调查集中在三个膜结合酶，VLCAD，ACAD 9，和TFP，和ETF与其电子转移合作伙伴，包括AD，二甲基甘氨酸脱氢酶（DD），和ETF-QO的相互作用。DD在胆碱代谢中起作用，并且不是AD家族的成员，但向ETF提供电子。具体目标是：2）VLCAD的研究，包括a）临床突变的研究，和B）通过EPR光谱确定VLCAD在线粒体膜上的取向以及与TFP和ETF的相互作用; 3）研究ACAD 9以确定其在线粒体复合物I组装中的独特作用的生物化学/结构基础;研究了ETF的电畴运动及其与三种典型给电子体的相互作用（中链酰基-CoA脱氢酶、VLCAD和DD）和其电子受体ETF-QO。

DESCRIPTION (provided by applicant): Fatty acid β-oxidation is the major energy-producing process in the liver, heart, and muscle. It is carried out by a series of four reactions that successively cleave acetyl-CoA from fatty acyl-CoA. The rate of this process can be altered by diet (fed/fasting), physiological status (pregnancy), or diseases (diabetes). The first of the four reactions in this process is initiated by a family of flavoproteins, acyl-CoA dehydrogenases (ADs). Electron transfer from ADs to the mitochondrial OXPHOS chain is catalyzed by electron transfer flavoprotein (ETF) and the membrane-bound ETF-ubiquinoneoxidoreductase (ETF-QO). There are at least seven soluble ADs for catalyzing short chain acyl-CoAs, and two membrane-bound ADs specific for long chain fatty acyl-CoAs, very long chain AD (VLCAD) and ACAD9. The three remaining reactions of β-oxidation for long chain fatty acids are carried out by the trifunctional protein (TFP), a membrane-bound multienzyme complex. Inborn errors of fatty acid oxidation have emerged as an increasing health problem and now represent the most common group of disorders identified through expanded newborn screening, affecting 2-3/1,000 babies born nationwide. These disorders present sudden infant death syndrome, cause cardiomyopathy, and are the most common cause of skeletal myopathy in older children and young adults. Recently ACAD9 has been shown to be essential for the assembly of Complex I, the largest and most complicated enzyme (~980 kDa with 45 subunits) among the five OXPHOS complexes. Very little is known concerning the mechanism of the assembly process of this important enzyme. Disorders of the mitochondrial OXPHOS system are the most common of inborn metabolic diseases, resulting in a wide variety of clinical phenotypes ranging from exercise intolerance to failure to thrive. We have determined the crystal structures of all but one of the soluble ADs, as well as one membrane-bound AD (VLCAD), ETF, and ETF-QO. The proposed investigations are focused on three membrane-bound enzymes, VLCAD, ACAD9, and TFP, and interactions of ETF with its electron transfer partners, including ADs, dimethylglycine dehydrogenase (DD), and ETF-QO. DD functions in choline metabolism and is not a member of the AD family, but donates electrons to ETF. Specific Aims are: 1) Structural studies of human TFP by X-ray crystallography to understand how its three distinct active sites communicate with each other; 2) Studies of VLCAD, including a) studies of clinical mutations, and b) to determine the orientation of VLCAD on the mitochondrial membrane and interactions with TFP and ETF by EPR spectroscopy; 3) studies of ACAD9 to determine the biochemical/structural basis for its unique role in mitochondrial Complex I assembly; and 4) to investigate the domain movement of ETF and its interactions with three representative electron donors (medium chain acyl-CoA dehydrogenase, VLCAD, and DD) and with its electron acceptor, ETF-QO.