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基，是重要的调控分子。革兰氏阴性杆菌大肠杆菌可以利用这些化合物作为唯一的碳和能量来源来支持生长，因此已经进化出一种高度特异性的系统来运输它们穿过细胞包膜。外源长链脂肪酸通过一个浓缩过程被吸收，这个浓缩过程与利用紧密耦合，并且至少回收外膜蛋白FadL （FadL基因的产物）和内膜相关的脂肪酰基辅酶a合成酶（FACS， fadD基因的产物）。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ATP水解激活长链脂肪酸，从而激活长链脂肪酸在细胞膜上的转运，这表明长链脂肪酸的转运过程对细胞的能量状态是有响应的。分析大肠杆菌长链脂肪酸转运系统功能特性的研究将确定脂肪酸结合转运和噬菌体T2结合所需的长链脂肪酸转运蛋白FadL内的结构域，确定脂肪酸酰基CoA合成酶的底物结合区域，确定脂肪酸酰基CoA合成酶作为长链脂肪酸转运装置组成部分的作用。假设FadL的氨基端近端结构域参与脂肪酸结合，膜结合结构域形成长链脂肪酸特异性通道。此外，我们认为FadL负责结合噬菌体T2的区域位于氨基末端近端，并且与长链脂肪酸结合域重叠。本研究结合了蛋白质分析（配体结合和亲和标记）和fadL基因的定向诱变以及表型分析来验证这些假设。在预测三级结构的基础上，提出脂肪酸酰基辅酶a合成酶含有两个重叠结构域，分别参与ATP和长链脂肪酸的结合。这一假设将通过脂肪酸和ATP亲和标记纯化酶的组合来验证；利用荧光光谱研究配体结合（脂肪酸和ATP）；以及结合表型和生理特性的fadD基因定向诱变。脂肪酸酰基辅酶a合成酶被认为可进入细菌内膜，并发挥促进外源长链脂肪酸硫酯化单向转运的作用。这将使用纯化的脂肪酰基辅酶a合成酶、由AfadD菌株制备的内膜囊泡和酶底物（脂肪酸、ATP和辅酶a）进行测试。细菌分子遗传系统是研究控制长链脂肪酸运输过程的一般和特定机制以及定义和表征该系统中蛋白质成分功能域的理想条件。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.