Acinetobactin and C-Glucosylated Enterobactin Siderophores

描述(由申请人提供)：第一个重点是在铁限制的微环境中，包括脊椎动物宿主，打开铁载体肠动蛋白的生物合成基因的肠杆菌菌株。肠杆菌素能与铁离子强烈结合，并被产生菌特异性摄取。一些致病的革兰氏阴性菌进一步阐述了肠杆菌素支架，以产生C-葡萄糖化形式，也被称为Salmochelin，因为他们在清除铁的伤寒沙门氏菌菌株中发现了这种物质。Salmochelin产生菌在Iro基因簇IroBCDEN的指导下定制Ent支架。我们将研究IroB进行C-糖基化的酶机制，并确定Ent支架上二羟基苯环的糖基化在多大程度上干扰哺乳动物宿主蛋白铁胆碱对铁载体的隔离。未能隔离修饰的肠杆菌素应该与细菌的致病性增加有关。第二个焦点是由革兰氏阴性呼吸道病原体鲍曼不动杆菌产生的铁载体acinobactin。铁载体的产生与毒力的增加相关。Acinetobactin具有所有三个已知的铁络合基，儿茶酚、噻唑啉和异羟甲酸酯，通过非核糖体多肽合成酶装配线构建到其骨架中。这六个基因编码铁载体合成酶装配线。我们计划大量生产每一种蛋白质，并评估以下预测用于装配线操作的不同寻常的特征：两个独立的腺化和硫代化结构域的作用，串联缩合结构域的环脱氢反应，铁载体成熟过程中链终止过程中异羟甲酸酯的形成。Salrnochelin和Acinetobactin生物合成酶的特性将是随后评估可能阻止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.