Biochemistry of Fatty Acid Uptake in Escherichia coli

长链脂肪酸（LCFA <$C12- C18！）穿越所述单元的 被高亲和力、可饱和、能量- 一个依赖过程，它需要at 至少两个基因，fadL和fadD。 fadL基因编码一个外 膜结合蛋白（FadL）介导跨膜转运 膜层 fadD基因编码酰基辅酶A合成酶， 激活脂肪酸的内膜相关酶 与运输相伴。 越来越多的证据表明， 低分子量周质脂肪酸结合蛋白和一种 内膜结合油酸结合蛋白也参与 在运输方面。 在拟议的研究中，定点，化学- 饱和和缺失突变的克隆fadL+基因将 用于区分与FadL相关的生化特性： LCFA结合、LCFA转运、噬菌体T2结合和热- 可修改性 这些研究将通过使用 亲和标记的长链脂肪酸以鉴定潜在的氨基酸 参与结合和转运的FadL的酸性残基， 表征结合和转运缺陷突变体。 酰基 将测定CoA合成酶基因（fadD）序列，然后 诱变实验，以评估结构和功能 这种酶的特性需要这个运输过程。 这项工作的目标是提供一个了解多久 链脂肪酸结合并穿过细胞膜， 大肠埃希菌 从广义上讲，这些实验的结果 将提供描述蛋白质分子基础的信息- 转运和脂肪酸活化过程中的脂肪酸相互作用 在代谢转化之前。

Long-chain fatty acids (LCFAs ıC12 - 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.