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. ***
