Structure and Function of Enzymes in Fatty Acid Oxidation

描述(申请人提供)：脂肪酸？-氧化是肝脏、心脏和肌肉的主要能量产生过程。它是通过一系列的四个反应来进行的，这些反应依次从脂肪酰基-辅酶A中裂解出乙酰-辅酶A。这一过程的速度可以通过饮食(饮食/禁食)、生理状态(怀孕)或疾病(糖尿病)来改变。四个中的第一个 这一过程中的反应是由一族黄素蛋白--酰基辅酶A脱氢酶(ADS)启动的。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ADS向线粒体OXPHOS链的电子传递。至少有7种可溶性ADS可催化短链酰基-COAS，2种膜结合型ADS专用于长链脂肪酰基-COAS、超长链AD(VLCAD)和ACAD9。长链脂肪酸的其余三个氧化反应是由三功能蛋白(TFP)完成的，TFP是一种膜结合多酶复合体。先天性脂肪酸氧化错误已经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发现的最常见的疾病组，影响到全国出生的1000名婴儿中的2-3名。这些疾病表现为婴儿猝死综合症，导致心肌病，是年龄较大的儿童和年轻人骨骼肌病的最常见原因。最近，ACAD9被证明是五个OXPHOS复合体中最大、最复杂的酶(~980 kDa，45个亚基)组装所必需的。关于这种重要酶的组装过程的机制，人们知之甚少。线粒体OXPHOS系统障碍是最常见的先天性代谢性疾病，导致从运动不耐受到无法茁壮成长的各种临床表型。我们已经确定了除一个外的所有可溶性ADS的晶体结构，以及一个膜结合AD(VLCAD)、ETF和ETF-QO。拟议的研究集中在三种膜结合酶，VLCAD，ACAD9和TFP，以及ETF与其电子转移伙伴ADS，二甲甘氨酸脱氢酶(DD)和ETF-QO的相互作用。DD在胆碱代谢中发挥作用，不是AD家族的成员，但向ETF捐赠电子。具体目标是：1)用X射线结晶学研究人TFP的结构，以了解其三个不同的活性部位是如何相互作用的；2)VLCAD的研究，包括a)临床突变的研究，b)通过EPR光谱确定VLCAD在线粒体膜上的定位以及与TFP和ETF的相互作用；3)ACAD9的研究，以确定其在线粒体复合体I组装中独特作用的生化/结构基础；以及4)研究ETF的结构域移动及其与三个具有代表性的电子供体(中链酰基辅酶A脱氢酶、VLCAD和DD)及其电子受体ETF-QO的相互作用。 与公共健康相关：人体内的所有细胞都需要能量来维持其结构完整性和履行其重要功能。这些能量的主要部分来自心脏、肝脏和肌肉细胞中脂肪酸的氧化，这一过程中的不平衡会导致疾病状态，如肥胖和糖尿病。更好地了解这些酶的功能将为诊断以及设计和开发用于治疗代谢紊乱的新型抑制剂或激动剂提供洞察力。

DESCRIPTION (provided by applicant): Fatty acid ?-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 ?- 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. PUBLIC HEALTH RELEVANCE: All cells in the human body need energy to maintain their structural integrity and to perform their vital functions. The major proportion of this energy comes from the oxidation of fatty acids in heart, liver, and muscle cells and an imbalance in this process results in disease states, such as obesity and diabetes. A better understanding of how these enzymes function will provide insight into diagnosis and the design and development of novel inhibitors or agonists for the treatment of metabolic disorders.