Biochemistry of Fatty Acid Uptake in Escherichia Coli

点击翻译按钮获取中文摘要

Long-chain fatty acids (LCFA) traverse the cell envelope of Escherichia coli by a high-affinity, saturable, energy-dependent process. This uptake system requires the functional activities of at least two genes, fadL and fadD, so that LCFA can be used as a sole carbon and energy source. The fadL gene codes for an outer membrane-bound long-chain fatty acid binding protein, FadL, that is required for the transport of LCFAs into the cell. The fadD gene codes for acyl CoA synthetase, an enzyme presumed to be associated with the inner membrane. The kinetics of LCFA binding to whole cells blocked for fatty acid utilization will be determined in an effort to further define the role of FadL. In particular, the temperature, pH optima, and fatty acid chain length specificity of fatty acid binding will be determined. The cloned fadL+ gene will be mutagenized in vitro using hydroxylamine and in vivo by cycling through mutator strains to generate mutants in fadL that will be used in defining the functional domains of FadL. The fatty acid binding domains will be identified using affinity-labelled LCFAs coupled with peptide sequencing. Collectively, the data generated from this project will be used to develop a model correlating the functional domains of FadL with the linear amino acid sequence. In more broad terms, the information gleaned from these experiments will provide information describing the molecular basis of protein- fatty acid interaction at the cell surface. This description, therefore, may be applied to more complex cell types in evaluating how fatty acids traverse the membrane.