Acinetobactin and C-Glucosylated Enterobactin Siderophores

描述（由申请人提供）：第一个重点是肠杆菌菌株，该菌株在铁受限的微环境中（包括脊椎动物宿主）开启铁载体肠杆菌的生物合成基因。肠obactin能与铁紧密结合，并被产菌特异性吸收。一些致病性革兰氏阴性菌进一步细化肠杆菌蛋白支架，以制造c -糖基化形式，也被称为从铁清除沙门氏菌伤寒菌株中发现的盐切菌素。产蝶呤的细菌在Iro基因簇IroBCDEN的指导下定制Ent支架。我们将研究IroB对C-糖基化的酶促机制，并确定Ent支架的二羟基苯环的糖基化在多大程度上干扰了哺乳动物宿主蛋白铁苷对铁载体的隔离。不能隔离修饰的肠杆菌应该与细菌的致病性增加有关。第二个重点是革兰氏阴性呼吸道病原体鲍曼不动杆菌产生的铁载体不动杆菌素。铁载体的产生与毒力的增加有关。不动乳酸菌蛋白具有所有三种已知的铁螯合基团，儿茶酚，噻唑啉和羟酸酯，通过非核糖体肽合成酶装配线构建到其骨架中。6个基因BasABCDEF编码铁载体合成酶装配线。我们计划过量生产每种蛋白质，并评估流水线操作预测的以下不寻常特征：两个独立的腺苷化和硫酰化结构域的作用，串联缩合结构域的环脱水，铁载体成熟过程中链终止过程中的羟酸盐形成。salnnochelin和acinetoactin生物合成酶的表征将是后续评价可能阻断铁载体产生的酶抑制剂的基础。

DESCRIPTION (provided by applicant): The first focus is on enterobacterial strains that turn on biosynthetic genes for the siderophore enterobactin in microenvironments where iron is limiting, including vertebrate hosts. Enterobactin binds ferric iron avidly and is subjected to specific uptake by the producing bacteria. Some pathogenic gram negatives further elaborate the enterobactin scaffold to make C-glucosylated forms, also known as salmochelins from their discovery in iron-scavenging salmonella typhi strains. Salmochelin-producing bacteria tailor the Ent scaffold under direction of the Iro gene cluster IroBCDEN. We will study the enzymatic mechanism for C- glucosylation by IroB and determine to what extent the glucosylation of the dihydroxybenzene rings of the Ent scaffold interfere with sequestration of the siderophore by the mammalian host protein siderocalin. Failure to sequester the modified enterobactins should correlate with increased pathogenicity of the bacteria. The second focus is on the production of the siderophore acinteobactin by the gram negative respiratory pathogen Acinetobacter baumanii. Siderophore production correlates with increased virulence. Acinetobactin has all three known-iron chelating groups, catechol, thiazoline, and hydroxamate, built into its skeleon by a nonribosomal peptide synthetase assembly line. The six genes BasABCDEF encode the siderophore synthetase assembly line. We plan to overproduce each protein and evaluate the following unusual featiures predicted for assembly line ooperations: action of two free standing adenylation and thiiolation domains, cyclodehdration by tandem condensation domains, hydroxamate formation during chain termination in siderophore maturation. Characterization of the salrnochelin and acinetobactin biosynthesizing enzymes will be the foundation for subsequent evaluation of enzyme inhibitors that might block siderophore production.