Biochemistry of Fatty Acid Uptake in Escherichia Coli

大肠杆菌摄取脂肪酸的生物化学

• 批准号: 8811714
• 负责人: Paul Black
• 金额: $ 21.46万
• 依托单位: The University of Tennessee, Memphis - The Health Science Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8811714&HistoricalAwards=false
• 关键词: Biochemistry; Fatty; Acid; Uptake; Escherichia

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.

长链脂肪酸（LCFA）穿过大肠杆菌的细胞被膜 大肠杆菌通过一个高亲和力，饱和，能量依赖的过程。 这 摄取系统需要至少两个基因的功能活性， fadL和fadD，使LCFA可以作为唯一的碳和能源 源头 fadL基因编码外膜结合长链 脂肪酸结合蛋白，FadL，这是运输所需的 LCFA进入细胞。 fadD基因编码酰基辅酶A合成酶， 推测与内膜有关的酶。 LCFA与全细胞结合的动力学被脂肪酸阻断 利用率将被确定，以进一步定义 的FadL。 特别地，温度、最适pH和脂肪酸 将测定脂肪酸结合的链长特异性。 的 克隆的fadL+基因将在体外使用羟胺诱变， 在体内通过在突变菌株中循环以产生fadL中的突变体 其将用于定义FadL的功能结构域。 的 脂肪酸结合域将使用亲和标记的 LCFA结合肽测序。 总的来说，数据 从这个项目中产生的将用于开发一个模型， 具有线性氨基酸序列的FadL的功能结构域。 从更广泛的意义上说，从这些实验中收集到的信息 将提供描述蛋白质分子基础的信息- 脂肪酸在细胞表面的相互作用。 这种描述， 因此，可以应用于更复杂的细胞类型， 脂肪酸穿过膜。