Biochemistry of Fatty Acid Transport in Escherichia coli

在所有生物体中，脂肪酸（FA）及其衍生物是膜的组成部分，是代谢能量的来源，是调节代谢的效应分子。这项研究是关于长链脂肪酸（C14-C18）进入细胞的运输，然后在代谢之前将其酶转化为辅酶A硫酯。这些FAs通过一种特定的、能量依赖的过程穿过大肠杆菌的细胞包膜，该过程需要外膜结合FA结合蛋白FadL和内膜相关酰基辅酶a合成酶（ACS）。ACS激活伴随转运的FAs，并导致细胞内FA的净积累，对抗浓度梯度。控制FadL介导的长链FA跨外膜运输的过程将通过以下方法确定：1 .利用有限的蛋白质水解和蛋白质修饰评估FadL的拓扑结构；2 .利用亲和标记的长链脂肪酸9-对氮杂氧壬酸（3H-APNA）确定FadL内FA结合袋。通过使用亲和标记配体azido- （32P ATP和3H-APNA）分别定义ACS内ATP和FA结合域，以及在涉及CoA和/或FA结合的特定位点上诱变fadD基因，将评估酰基CoA合成酶对长链FA转运的贡献。研究也正在进行，以确定膜结合（FadL）和可溶性蛋白组分之间的蛋白质-蛋白质相互作用，使用远西方分析，并通过谷胱甘肽s -转移酶（GST）和组氨酸融合蛋白进行实验。可溶性蛋白成分可能与FadL相互作用。胞膜内的H+/FA共转运体、胞浆内的酰基辅酶a脱氢酶和酰基辅酶a结合蛋白可能与ACS特异性结合。在从细菌到人类的活细胞中，膳食脂肪（或脂肪酸；FAs）是支持生长的重要能量来源。这个研究项目解决了长链FAs如何穿过细胞膜进入细胞以产生能量的问题。在大肠杆菌中，长链脂肪酸被一种酶系统吸收，该酶系统需要一种外膜结合的脂肪酸结合和转运蛋白（FadL）和一种内膜相关酶（CoA合成酶；ACS）。使用大肠杆菌进行这些研究是理想的，因为可以利用生物化学、分子生物学和分子遗传学来回答有关长链FAs转运的基本问题。工作将定义FadL中对FA运输至关重要的区域，从而作为理解蛋白质与FA之间特定相互作用的范例。此外，还将确定酰基辅酶a合成酶内结合长链FAs和ATP的区域，从而提供该酶在FA转运中的作用的信息。最后，正在进行研究以确定运输系统中膜结合蛋白（FadL）和可溶性蛋白组分之间的蛋白-蛋白相互作用。这些研究对于进一步定义转运系统，以及通过确定与FadL和ACS形成的特异性细胞质/膜蛋白关联来发生FA转运具有重要意义。＊＊＊

9506059 Black In all organisms, fatty acids (FA) and their derivatives are components of membranes, are sources of metabolic energy, and are effector molecules that regulate metabolism. This research is on the transport of long-chain fatty acids (C14-C18) into the cell, followed by their enzymatic conversion to coenzyme A thioesters prior to metabolism. These FAs traverse the cell envelope of Escherichia coli by a specific, energy-dependent process that requires the outer membrane-bound FA binding protein FadL and the inner membrane associated acyl CoA synthetase (ACS). ACS activates FAs concomitant with transport and results in net FA accumulation in the cell against a concentration gradient. Processes that govern FadL-mediated long-chain FA transport across the outer membrane will be determined by i evaluating the topology of FadL using limited proteolysis and protein modification and ii defining the FA binding pocket within FadL using the affinity labeled long-chain fatty acid 9-p-azidophenoxy nonanoic acid (3H-APNA). The contribution of acyl CoA synthetase to long-chain FA transport will be evaluated by i defining the ATP and FA binding domains within ACS using the affinity labeled ligands azido- (32P ATP and 3H-APNA, respectively, and ii mutagenesis of the fadD gene at specific sites involved in CoA and/or FA binding. Studies are also being conducted to define protein-protein interactions between the membrane-bound (FadL) and soluble protein components of this transport system using far Western analyses, and by performing experiments with glutathione S-transferase (GST) and histidine fusion proteins. Soluble protein components may interact with FadL. The H+/FA cotransporter in the inner cell membrane, and acyl CoA dehydrogenase and acyl CoA binding protein in the cell cytosol may bind specifically with ACS. %%% In living cells from bacteria to man, dietary fats (or fatty acids; FAs) are important sources of energy to support growth. This research project addresses the qu estion of how long-chain FAs are transported across the membrane into the cell to be used to produce energy. In the bacterium, Escherichia coli, long-chain FAs are taken up by an enzyme system that requires an outer membrane-bound FA binding and transport protein (FadL) and an inner membrane associated enzyme (CoA synthetase; ACS). The use of E. coli for these studies is ideal as basic questions can be answered concerning transport of long-chain FAs using biochemistry, molecular biology and molecular genetics. Work will define regions within FadL that are essential in FA transport, and will thus serve as a paradigm in understanding specific interactions between proteins and FAs. In addition, regions within acyl CoA synthetase that bind long-chain FAs and ATP will be defined, thus providing information on the role of this enzyme in FA transport. Lastly, studies are being conducted to define protein-protein interactions between the membrane-bound (FadL) and soluble protein components of the transport system. These studies will be important in further defining the transport system and by identifying specific cytosolic/membraneous protein associations that develop with FadL and with ACS for FA transport to occur. ***