Biochemistry of Fatty Acid Uptake in Escherichia Coli

长链脂肪酸(LCFA)通过一种高亲和力的、可饱和的、能量依赖的过程穿过大肠杆菌的细胞膜。这种摄取系统需要至少两个基因Fad1和FadD的功能活性，这样LCFA才能被用作唯一的碳和能源。Fadl基因编码一种外膜结合的长链脂肪酸结合蛋白Fadl，它是将低密度脂蛋白脂肪酸运输到细胞内所必需的。FADD基因编码酰基辅酶A合成酶，一种被认为与内膜相关的酶。为了进一步确定Fadl的作用，将确定LCFA与因利用脂肪酸而被阻断的整个细胞结合的动力学。特别是，将确定脂肪酸结合的温度、最适pH和脂肪酸链长特异性。克隆的Fadl+基因将在体外用羟胺进行诱变，并在体内通过在突变株之间循环产生突变体，用于确定Fadl的功能结构域。脂肪酸结合域将使用亲和标记的LCFAs结合多肽测序来鉴定。总的来说，这个项目产生的数据将被用来开发一个将FADL的功能域与线性氨基酸序列相关联的模型。更广泛地说，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，在评估脂肪酸如何穿过细胞膜时，这一描述可能适用于更复杂的细胞类型。

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.