MOLECULAR ANALYSIS OF FERRIC ENTEROBACTIN TRANSPORT

所需代谢物在革兰氏阴性菌中的主动运输 细胞成分之间复杂相互作用的结果 定位于外膜、胞质膜和周质 太空。我们研究计划的总体目标是开发一种 可以在其中探测这些相互作用的模型传输系统。在……里面 具体地说，这项提案旨在划定结构和 涉及的三种特异性大肠杆菌蛋白的功能特性 邻苯二酚铁载体ENTB的生物合成(EntD) (肠球蛋白)和铁载体介导的摄取(Fepa，Fes)。 具有重要营养价值的微量元素铁。人类的结构基因 已分离出这三个组分，并测定了它们的核苷酸序列 下定决心。蛋白质产品已被鉴定，并在接下来的病例中 纯净的。与蛋白质一起进行的体内外诱变程序 将使用生物化学方案来评估结构和 功能特征：(1)外膜受体(FebA)结合 其三种特定配体的结合部位：肠杆菌素、结肠素D和结肠素 粘菌素B；(2)细胞质酶Fes，它负责 当铁载体复合体进入细胞时，从铁载体复合体中去除铁； 和(3)膜相关生物合成酶EntD，包括 其与其他组分(Ente、Entf)的分子结合性质 和EntG)，其 新合成的铁载体在胞外释放中的潜在作用 分子。 除了作为模型的基本科学兴趣外，它还具有很高的亲和力 运输系统，对铁运输机制的理解是 重要的是，在所有被研究的生物体中，从细菌到人类， 机体的代谢铁状态调节着营养的程度 铁的吸收和铁已经被证明是 人和人传播革兰氏阴性杆菌感染的病理生理学 动物。希望该系统能够提供一种定义 铁运输的基本特征与同类产品的比较 来自其他革兰氏阴性病原体的系统，以及对哪些系统有效 未来的治疗可能是有针对性的。

Active transport of required metabolites in gram negative organisms is the result of complex interactions between cellular constituents localized in the outer and cytoplasmic membranes and the periplasmic space. The overall objectives of our research plan are to develop a model transport system in which these interactions may be probed. In particular, this proposal is designed to delineate structural and functional features of three specific Escherichia coli proteins involved in the biosynthesis (EntD) of the catechol siderophore enterobactin (enterochelin) and the siderophore-mediated uptake (FepA, Fes) of the nutritionally important trace element, iron. The structural genes for these three components have been isolated and their nucleotide sequences determined. The protein products have been identified and in come cases purified. In vitro and in vivo mutagenesis procedures along with protein biochemistry protocols will be employed to evaluate structural and functional features of: (1) the outer membrane receptor (FebA) binding sites for its three specific ligands, ferric enterobactin, colicin D and colicin B; (2) the cytoplasmic enzyme Fes, which is responsible for removing iron from the siderophore complex upon its entry into the cell; and (3) the membrane-associated biosynthetic enzyme EntD, including the nature of its molecular association with the other components (EntE, EntF and EntG) of the multienzyme complex enterobactin synthetase, and its potential role in extracellular release of newly synthesized siderophore molecules. Aside from its basic scientific interest as a model high affinity transport system, an understanding of the mechanisms of iron transport is significant is that in all organisms studied, from bacterial to man, the metabolic iron state of the organism regulates the degree of nutritive iron absorption and iron has been documented as an important factor of the pathophysiology of disseminating gram negative infections in man and animals. It is hoped that this system may present a model defining the essential characteristics of iron transport for comparison with similar systems from other gram negative pathogens and against which effective future therapies may be targeted.