Biochemistry of FAtty Acid Transport In Escherichia Coli

长链脂肪酸(Cx4-CLS)是能量产生和大分子生物合成的重要底物，也是重要的调节分子。革兰氏阴性菌大肠杆菌可以利用这些化合物作为唯一的碳和能源来支持生长，从而进化出一种高度特异的系统，用于它们在细胞膜上的运输。外源长链脂肪酸是通过一种与利用相耦合的浓缩过程被摄取的，至少产生外膜蛋白FADL(FADL基因产物)和内膜相关脂肪酰辅酶A合成酶(FACS；FADD基因产物)。FADL以高亲和力结合外源长链脂肪酸，促进其跨外膜运输。FACS通过三磷酸腺苷的水解激活伴随着跨内膜运输的长链脂肪酸，证明了长链脂肪酸的运输过程是对细胞的通电状态作出反应。通过对大肠杆菌中长链脂肪酸转运系统的功能特性的分析，将确定脂肪酸结合和转运所需的长链脂肪酸转运蛋白FAD1中的结构域和噬菌体T2结合的结构域，定义脂肪酰辅酶A合成酶的底物结合区，并确定脂肪酰辅酶A合成酶作为长链脂肪酸运输装置的组成部分的作用。FADL的氨基末端近端结构域被认为参与了脂肪酸的结合，膜结合区形成了长链脂肪酸特异性通道。此外，Fad1负责与噬菌体T2结合的区域位于氨基末端，并与长链脂肪酸结合区重叠。这项研究结合了蛋白质分析(配体结合和亲和标记)和Fadl基因的定向突变，并结合表型分析来验证这些假说。根据预测的三级结构，脂肪酰辅酶A合成酶含有两个重叠的结构域，分别参与三磷酸腺苷和长链脂肪酸的结合。这一假设将通过对纯化的酶进行脂肪酸和ATP亲和标记，使用荧光光谱进行配体结合研究(脂肪酸和ATP)，以及结合表型和生理特征的FADD基因的定向突变来验证。脂肪酰辅酶A合成酶被认为是通过外源长链脂肪酸的硫代酯化作用进入细菌内膜，促进其单向转运。这将使用纯化的脂肪酰辅酶A合成酶、从AfadD菌株制备的内膜小泡和酶底物(脂肪酸、ATP和辅酶A)进行测试。细菌分子遗传系统非常适合研究控制长链脂肪酸运输过程的一般和特定机制，并定义和表征该系统中蛋白质组分的功能域。这项工作专门研究了蛋白质-脂肪酸在运输过程中相互作用的分子机制，因此将作为理解这一过程的范例。

Biochemistry of Fatty Acid Transport in Escherichia coli BLACK, Paul N.ABSTRACTBLACKLong-chain fatty acids (C x4 - Cls) are important substrams for energy production and macromolecular biosynthesis and are important regulatory molecules. The gram negative bacterium Escherichia coli can utilize these compounds as a sole carbon and energy source to support growth and thus has evolved a highly specific system for their transport across the cell envelope. Exogenous long-chain fatty acids are taken up by a concentrative process that is tigh~y coupled to utilization and reclulres at least the outer membrane protein FadL (product of the fadL gene) and the inner membrane associated fatty acyl CoA synthetase (FACS; product of the fadD gene). FadL binds exogenous long-chain fatty acids with high affinity and facilitates their transport across the outer membrane. FACS activates long-chain fatty acids concomitant with transport across the inner membrane by a process that proceeds through the hydrolysis of ATP demonstrating that process of long-chain fatty acid transport is responsive to the energized state of the cell. The research analyzing the functional properties of the long-chain fatty acid transport system in E. coli will define domains within the long-chain fatty acid transport protein FadL required for fatty acid binding and transport and bacteriophage T2 binding, define the substrate binding regions of fatty acyl CoA synthetase and define the role of fatty acyl CoA synthetase as a component of the long-chain fatty acid transport apparatus. The amino-terminal proximal domain of FadL is hypothesized to be involved in fatty acid binding and that the membrane-bound domain forms a long-chain fatty acid specific channel. Furthermore, it is suggested that the region of FadL responsible for binding bacteriophage T2 is amino-terminal proximal and overlaps the long-chain fatty acid binding domain. This research employs a combination of protein analyses (ligand binding and affinity labeling) and directed mutagenesis of the fadL gene coupled with phenotypic analyses to test these hypotheses. On the basis of predicted tertiary structure, fatty acyl CoA synthetase is proposed to contain two overlapping domains which are involved in the binding of ATP and long-chain fatty acid respectively. This hypothesis will be tested using a combination of fatty acid and ATP affinity labeling of the purified enzyme; ligand binding studies (fatty acid and ATP) using fluorescence spectroscopy; and directed mutagenesis of the fadD gene coupled with phenotypic and physiological characterization. Fatty acyl CoA synthetase is thought to partition into the inner bacterial membrane and function to facilitate the unidirectional transport by thioesterification of exogenous long-chain fatty acids. This will be tested using purified fatty acyl CoA synthetase, inner membrane vesicles prepared from a AfadD strain, and enzyme substrates (fatty acid, ATP, and coenzymeA). The bacterial molecular-genetic system is ideally poised to investigate the general and specific mechanisms that govern the process of long-chain fatty acid transport and define and characterize the functional domains of the protein components in this system. This work specifically addresses the molecular mechanisms of protein-fatty acid interaction during the process of transport and thus will serve as a paradigm for understanding this process.