MOLECULAR ANALYSIS OF FERRIC ENTEROBACTIN TRANSPORT

革兰氏阴性菌中所需代谢物的主动转运是 细胞成分之间复杂相互作用的结果 定位于外膜、细胞质膜和周质 空间 我们的研究计划的总体目标是开发一个 模型运输系统，其中可以探测这些相互作用。 在 特别是，这项建议旨在界定结构和 三种特异性大肠杆菌蛋白的功能特征 在儿茶酚铁载体肠杆菌素的生物合成（EntD）中 （肠螯合素）和铁载体介导的摄取（FepA，Fes） 重要的微量营养元素铁 结构基因 这三种组分已被分离，它们的核苷酸序列 测定蛋白质产物已被鉴定，在某些情况下， 提纯 体外和体内诱变程序沿着蛋白质 生物化学方案将用于评估结构和 功能特点：（1）外膜受体（FebA）结合 它的三个特异性配体，铁肠杆菌素，大肠杆菌素D和 大肠杆菌素B;（2）细胞质酶Fes，负责 在铁载体复合物进入细胞时从铁载体复合物中除去铁; 和（3）膜结合生物合成酶EntD，包括 其与其他组分（EntE，EntF）分子缔合的性质 和EntG）的多酶复合物肠杆菌素合成酶，及其 新合成的铁载体在细胞外释放中的潜在作用 分子。 除了作为一个模型的基本科学兴趣之外， 运输系统，铁运输机制的理解是 重要的是，在所有研究的生物体中，从细菌到人类， 生物体的铁代谢状态调节营养的程度， 铁的吸收和铁已被证明是一个重要的因素， 在人类中传播革兰氏阴性感染的病理生理学 动物 希望该系统可以提供一个定义 铁运输的基本特征与类似的 系统从其他革兰氏阴性病原体，并针对其有效 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.