Biochemistry of Fatty Acid Transport in Escherichia coli

大肠杆菌脂肪酸转运的生物化学

• 批准号: 9506059
• 负责人: Paul Black
• 金额: $ 10万
• 依托单位: The University of Tennessee, Memphis - The Health Science Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-11-01 至 1996-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506059&HistoricalAwards=false
• 关键词: Biochemistry; Fatty; Acid; Transport; Escherichia

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. ***

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促转化为辅酶A硫酯。 这些FA通过一个特定的能量依赖性过程穿过大肠杆菌的细胞被膜，该过程需要外膜结合的FA结合蛋白FadL和内膜相关的酰基CoA合成酶（ACS）。 ACS激活FA伴随着运输，并导致细胞中的净FA积累，对抗浓度梯度。 控制FadL介导的长链FA跨外膜转运的过程将通过i使用有限的蛋白水解和蛋白修饰评估FadL的拓扑结构和ii使用亲和标记的长链脂肪酸9-对叠氮苯氧基壬酸（3 H-APNA）定义FadL内的FA结合口袋来确定。 酰基CoA合成酶对长链FA转运的贡献将通过i分别使用亲和标记的配体叠氮基-β 2 P ATP和3 H-APNA定义ACS内的ATP和FA结合结构域，和ii在涉及CoA和/或FA结合的特定位点诱变fadD基因来评价。还正在进行研究，以确定蛋白质-蛋白质之间的相互作用的膜结合（FadL）和可溶性蛋白质组分的运输系统使用远Western分析，并通过实验谷胱甘肽S-转移酶（GST）和组氨酸融合蛋白。 可溶性蛋白组分可与FadL相互作用。 细胞内膜上的H+/FA共转运蛋白、细胞质中的酰基CoA脱氢酶和酰基CoA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; FA）是支持生长的重要能量来源。 这个研究项目解决了长链脂肪酸如何穿过细胞膜进入细胞以产生能量的问题。 在细菌大肠杆菌中，长链FA被需要外膜结合FA结合和转运蛋白（FadL）和内膜相关酶（CoA合成酶; ACS）的酶系统吸收。 利用E.大肠杆菌的这些研究是理想的，因为基本的问题，可以回答有关运输的长链脂肪酸使用生物化学，分子生物学和分子遗传学。 工作将定义在FA运输中必不可少的FadL内的区域，并将因此作为理解蛋白质和FA之间特定相互作用的范例。 此外，酰基辅酶A合成酶内的区域，结合长链脂肪酸和ATP将被定义，从而提供信息的作用，这种酶在脂肪酸运输。 最后，正在进行研究，以确定蛋白质-蛋白质之间的相互作用的膜结合（FadL）和可溶性蛋白质组分的运输系统。 这些研究将是重要的，在进一步定义的运输系统，并确定特定的胞质/膜蛋白协会，发展与FadL和ACS的FA运输发生。 ***