Long-chain fatty acids (C x4 - Cls) are important substrams for energy production and macromolecular biosynthesis and are important regulatory molecules. The gram negative bacterium Escherichia coli can utilize these compounds as a sole carbon and energy source to support growth and thus has evolved a highly specific system for their transport across the cell envelope. Exogenous long-chain fatty acids are taken up by a concentrative process that is tigh~y coupled to utilization and reclulres at least the outer membrane protein FadL (product of the fadL gene) and the inner membrane associated fatty acyl CoA synthetase (FACS; product of the fadD gene). FadL binds exogenous long-chain fatty acids with high affinity and facilitates their transport across the outer membrane. FACS activates long-chain fatty acids concomitant with transport across the inner membrane by a process that proceeds through the hydrolysis of ATP demonstrating that process of long-chain fatty acid transport is responsive to the energized state of the cell. The research analyzing the functional properties of the long-chain fatty acid transport system in E. coli will define domains within the long-chain fatty acid transport protein FadL required for fatty acid binding and transport and bacteriophage T2 binding, define the substrate binding regions of fatty acyl CoA synthetase and define the role of fatty acyl CoA synthetase as a component of the long-chain fatty acid transport apparatus. The amino-terminal proximal domain of FadL is hypothesized to be involved in fatty acid binding and that the membrane-bound domain forms a long-chain fatty acid specific channel. Furthermore, it is suggested that the region of FadL responsible for binding bacteriophage T2 is amino-terminal proximal and overlaps the long-chain fatty acid binding domain. This research employs a combination of protein analyses (ligand binding and affinity labeling) and directed mutagenesis of the fadL gene coupled with phenotypic analyses to test these hypotheses. On the basis of predicted tertiary structure, fatty acyl CoA synthetase is proposed to contain two overlapping domains which are involved in the binding of ATP and long-chain fatty acid respectively. This hypothesis will be tested using a combination of fatty acid and ATP affinity labeling of the purified enzyme; ligand binding studies (fatty acid and ATP) using fluorescence spectroscopy; and directed mutagenesis of the fadD gene coupled with phenotypic and physiological characterization. Fatty acyl CoA synthetase is thought to partition into the inner bacterial membrane and function to facilitate the unidirectional transport by thioesterification of exogenous long-chain fatty acids. This will be tested using purified fatty acyl CoA synthetase, inner membrane vesicles prepared from a AfadD strain, and enzyme substrates (fatty acid, ATP, and coenzymeA). The bacterial molecular-genetic system is ideally poised to investigate the general and specific mechanisms that govern the process of long-chain fatty acid transport and define and characterize the functional domains of the protein components in this system. This work specifically addresses the molecular mechanisms of protein-fatty acid interaction during the process of transport and thus will serve as a paradigm for understanding this process.
