Biochemistry of Fatty Acid Uptake in Escherichia coli

长链脂肪酸(LCFAsıC12-C18！)通过一个高亲和力的、可饱和的、能量依赖的过程穿越大肠杆菌的细胞膜，该过程至少需要两个基因Fad1和FadD的功能活性。Fadl基因编码一种外膜结合蛋白(Fadl)，介导跨膜层的运输。FADD基因编码酰基辅酶A合成酶，这是一种内膜相关酶，可以激活伴随运输而来的脂肪酸。越来越多的证据表明，低分子量的周质脂肪酸结合蛋白和内膜结合的油酸结合蛋白也参与转运。在拟议的研究中，克隆的Fadl+基因的定点突变、化学饱和突变和缺失突变将用于区分与Fadl相关的生化特性：LCFA结合、LCFA转运、噬菌体T2结合和热修饰。这些研究将通过使用亲和标记的长链脂肪酸来加强，以确定参与结合和运输的FADL的潜在氨基酸残基，并表征结合和运输缺陷的突变体。将确定酰辅酶A合成酶基因(FADD)的序列，然后进行诱变实验，以评估该酶在这一运输过程中所需的结构和功能特征。这项工作的目标是提供一个了解有多长的链脂肪酸结合和穿越大肠杆菌的细胞膜。概括地说，这些实验的结果将提供描述蛋白质-脂肪酸在运输过程中相互作用和代谢转化前脂肪酸激活的分子基础的信息。

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.