Biochemistry of Fatty Acid Uptake in Escherichia Coli

9405803黑色脂肪酸是细胞膜的基本成分，是新陈代谢能量的重要来源。这个项目的重点是将长链脂肪酸转移到细胞内，然后在代谢利用之前将其酶转化为辅酶A硫代酯。在大肠杆菌中，这两个过程被认为是联系在一起的，因此长链脂肪酸的酯化提供了运输的动力。这些化合物通过一个高亲和力的、可饱和的、能量依赖的过程穿过细菌细胞膜，这个过程需要外膜结合的脂肪酸结合和运输蛋白Fadl(Fadl基因的产物)和内膜相关的酰基辅酶A合成酶(FADD基因的产物)。酰基辅酶A合成酶激活伴随着运输的脂肪酸，从而导致它们在细胞内相对于浓度梯度的净积累。FADL和FADD基因已被克隆和测序，并对其各自的基因产物进行了纯化和鉴定。在这项工作中，Fadl的拓扑结构将通过分离外膜的部分蛋白分解和多肽纯化和测序来定义。参与脂肪酸结合的FADL中的特定氨基酸残基将通过化学修饰和定点突变来确定。FADL内脂肪酸结合口袋内的氨基酸残基将通过亲和标记进行修饰，随后进行蛋白质分解、多肽纯化和标记氨基酸的鉴定。这将专门定义FADL中组成脂肪酸结合口袋的氨基酸，从而为利用FADL基因的突变进行更详细的研究奠定基础。总的来说，这些研究将定义Fadl如何跨越外膜，以及哪些特定的氨基酸残基位于脂肪酸结合口袋中。酰辅酶A合成酶在外源长链脂肪酸运输中的作用也将被进一步评估。来自大肠杆菌的酰基辅酶A合成酶与真核生物的合成酶有相当大的相似性，特别是在该酶假定与ATP结合的区域。酰辅酶A合成酶结构基因的定点突变将被用来定义该酶的这一区域是否代表ATP结合域。%脂肪酸是脂类的分子构件。因此，它们是细胞膜的重要组成部分，也是代谢能量的重要来源。根据细胞类型的不同，细胞所需的脂肪酸可以由细胞本身合成，和/或从周围环境中摄取。这个项目试图了解细胞从周围环境中专门吸收脂肪酸的生化机制，使用常见的模式细菌大肠杆菌作为模型系统。将结合使用遗传和生化方法。这些实验的结果有望扩大我们对脂肪酸以及一般大分子如何特定地跨细胞膜运输的理解。这类信息对于理解细胞的功能是至关重要的。不难看出，这种信息如何在下游应用到许多特别感兴趣的不同领域；例如，微生物生理生态学、药理学和动物或人类营养。***

9405803 Black Fatty acids are essential components of cellular membranes and are important sources of metabolic energy. This project focuses on the transport of long-chain fatty acids into cells followed by their enzymatic conversion to coenzyme A thioesters prior to metabolic utilization. In Escherichia coli, these two processes are postulated to be linked such that the esterification of long-chain fatty acids provides the driving force for transport. These compounds traverse the bacterial cell envelope by a high affinity, saturable, energy-dependent process that requires the outer membrane-bound fatty acid binding and transport protein FadL (product of the fadL gene) and the inner membrane associated acyl CoA synthetase (product of the fadD gene). Acyl CoA synthetase activates fatty acids concomitant with transport, thereby resulting in their net accumulation within the cell against a concentration gradient. The fadL and fadD genes have been cloned and sequenced and their respective gene products purified and characterized. In this work, the topology of FadL will be defined using partial proteolysis in isolated outer membranes and peptide purification and sequencing. Specific amino acid residues within FadL involved in fatty acid binding will be determined using chemical modification and site-directed mutagenesis. Amino acid residues within the fatty acid binding pocket within FadL will be modified using affinity labeling followed by proteolysis, peptide purification, and identification of the labeled amino acids. This will specifically define amino acids within FadL that comprise the fatty acid binding pocket and thus lay the groundwork for more detailed studies employing mutagenesis of the fadL gene. Collectively, these studies will define how FadL spans the outer membrane, and which specific amino acid residues sit within the fatty acid binding pocket. The role of acyl CoA synthetase in the transport of exogenous long-chain fatty acids will al so be further evaluated. Acyl CoA synthetase from E. coli has considerable similarity with its eukaryotic counterparts, particularly in a region of the enzyme postulated to bind ATP. Site-directed mutageneis of the acyl CoA synthetase structural gene will be used to define whether this region of the enzyme represents the ATP binding domain. %%% Fatty acids are the molecular building blocks of lipids. As such, they are essential components of cellular membranes, as well as important sources of metabolic energy. Depending on the cell type, the fatty acids needed by a cell may be either syntehsized by the cell itself, and/or taken up from the surrounding milieu. This project seeks to understand the biochemical mechanism whereby cells specifically take up fatty acids from their surroundings, using the common model bacterium, Escherichia coli, as a model system. A combination of genetic and biochemical approaches will be used. The results of these experiments are expected to expand our understanding of how fatty acids, and large molecules in general, are specifically transported across cell membranes. This kind of information is of fundamental importance to understanding how cells function. It is not difficult to see how this kind of information may have downstream application to a number of diverse areas of special interest; examples might include microbial physiological ecology, pharmacology, and animal or human nutrition. ***