Biochemistry of Fatty Acid Transport in Escherichia coli

9506059黑色在所有生物体中，脂肪酸(FA)及其衍生物是膜的组成部分，是代谢能量的来源，是调节新陈代谢的效应分子。这项研究是关于长链脂肪酸(C14-C18)进入细胞的运输，随后它们在代谢之前被酶转化为辅酶A硫代酯。这些FA通过一种特殊的、能量依赖的过程穿过大肠杆菌的细胞膜，该过程需要外膜结合的FA结合蛋白Fadl和内膜相关的酰辅酶A合成酶(ACS)。ACS激活伴随转运的FAs，并导致在细胞内相对于浓度梯度的净FA积累。调控Fadl介导的长链FA跨外膜转运的过程将通过I使用有限的蛋白质降解和蛋白质修饰来评估FADL的拓扑结构，以及II使用亲和力标记的长链脂肪酸9-对叠氮苯氧基壬酸(~3H-ApNA)来定义FADL内的FA结合口袋来确定。酰基辅酶A合成酶对长链FA转运的贡献将通过I使用亲和标记配体azido-(32P ATP和~3H-ApNA)在ACS内定义ATP和FA结合结构域以及II在涉及CoA和/或FA结合的特定位置对FADD基因的突变来评估。还在进行研究，以确定该运输系统的膜结合蛋白(FADL)和可溶性蛋白成分之间的蛋白质-蛋白质相互作用，并通过使用谷胱甘肽S转移酶(GST)和组氨酸融合蛋白进行实验。可溶性蛋白质组分可能与Fadl相互作用。细胞内膜的H+/FA共转运蛋白、胞浆中的酰基辅酶A脱氢酶和酰辅酶A结合蛋白可能与急性冠脉综合征特异结合。在从细菌到人类的活细胞中，膳食脂肪(或脂肪酸)是支持生长的重要能量来源。这项研究项目解决了长链脂肪酸如何跨膜进入细胞以产生能量的问题。在大肠杆菌中，长链FA被一个酶系统摄取，该系统需要一个外膜结合的FA结合和运输蛋白(FADL)和一个内膜相关酶(CoA合成酶；ACS)。使用大肠杆菌进行这些研究是理想的，因为利用生物化学、分子生物学和分子遗传学可以回答有关长链脂肪酸运输的基本问题。这项工作将定义Fadl内在FA运输中必不可少的区域，从而将作为理解蛋白质和FA之间特定相互作用的范例。此外，将定义酰基辅酶A合成酶中与长链FA和ATP结合的区域，从而提供关于该酶在FA运输中的作用的信息。最后，正在进行研究，以确定运输系统的膜结合蛋白(FADL)和可溶性蛋白成分之间的蛋白质-蛋白质相互作用。这些研究将对进一步确定转运系统和确定特定的胞浆/膜蛋白与FADL和ACS之间的联系以实现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. ***