Biochemistry of Fatty Acid Uptake in Escherichia Coli

长链脂肪酸（LCFA）通过高亲和力，饱和，能量依赖的过程穿过大肠杆菌的细胞包膜。这个吸收系统需要至少两个基因fadL和fadD的功能活动，这样LCFA才能作为唯一的碳和能量来源。fadL基因编码一种外膜结合的长链脂肪酸结合蛋白fadL，这是将LCFAs运输到细胞中所必需的。fadD基因编码酰基辅酶a合成酶，一种被认为与内膜有关的酶。为了进一步确定FadL的作用，将确定LCFA与被脂肪酸利用阻断的全细胞结合的动力学。特别是，温度、pH最优、脂肪酸链长对脂肪酸结合的特异性将被确定。克隆的fadL+基因将在体外用羟胺诱变，在体内通过突变菌株循环产生fadL突变体，这些突变体将用于定义fadL的功能域。脂肪酸结合结构域将使用亲和标记的LCFAs结合肽段测序进行鉴定。总的来说，从这个项目中产生的数据将用于开发一个将FadL的功能域与线性氨基酸序列相关联的模型。从更广泛的角度来看，从这些实验中收集的信息将提供描述细胞表面蛋白质-脂肪酸相互作用的分子基础的信息。因此，这种描述可以应用于更复杂的细胞类型，以评估脂肪酸如何穿过细胞膜。

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.