Long-chain fatty acids (LCFAs ?C12 - C18!) traverse the cell envelope of Escherichia coli by a high affinity, saturable, energy- dependent process that requires the functional activities of at least two genes, fadL and fadD. The fadL gene encodes an outer membrane-bound protein (FadL) that mediates transport across this membrane layer. The fadD gene encodes acyl CoA synthetase, an inner membrane associated enzyme that activates fatty acids concomitant with transport. There is increasing evidence that a low molecular weight periplasmic fatty acid binding protein and an inner membrane-bound oleic acid binding protein also participate in transport. In the proposed study, site-directed, chemical- saturation, and deletion mutagenesis of the cloned fadL+ gene will be used to distinguish biochemical properties associated with FadL: LCFA binding, LCFA transport, bacteriophage T2 binding, and heat- modifiability. These studies will be augmented by the use of affinity labeled long-chain fatty acids to identify potential amino acid residues of FadL involved in binding and transport, and to characterize binding- and transport-deficient mutants. The acyl CoA synthetase gene (fadD) sequence will be determined followed by mutagenesis experiments to evaluate the structural and functional features of this enzyme required for this transport process. The goal of this work is to provide an understanding of how long chain fatty acids bind to and traverse the cell envelope of Escherichia coli. In broad terms, the results of these experiments will provide information describing the molecular basis of protein- fatty acid interaction during transport and fatty acid activation prior to metabolic transformation.
