MOLECULAR ANALYSIS OF YERSINIABACTIN BIOSYNTHESIS

铁螯合铁载体和几种多肽抗生素 由非核糖体多肽合成酶复合物通过硫代模板组装而成 机制。铁载体Yersiniabactin，一个关键的毒力因子 耶尔森氏菌感染，既有启动生物合成的芳基-N-帽，又有 五元硫杂环(噻唑啉、噻唑烷类化合物 通过半胱氨酸的环化作用)来配位铁。这项研究 这里提出的建议涉及到确定负责 第一种酶促Yersiniabactin的生物合成及性质研究 涉及水杨酸、酰胺键形成的活化步骤 半胱氨酸残基和环化成的噻唑啉。我们建议 200kD高分子量蛋白2的YbtE及其结构域的纯化 并分析预期的酶活性，包括 翻译后磷酸肾素基化(PPANT)，水杨基-S-PPANT 酶负载、水杨基-环-S-酶的形成和环化为a 水杨基-噻唑啉-S-酶中间体。这些研究应该能破译 酶组装的逻辑不仅仅是毒力赋予的逻辑 铁载体Yersiniabactin和angibactin也是多肽 抗生素，如放线菌素、原始霉素和抗肿瘤药物 使用芳基N-帽仿和/或噻唑啉、恶唑啉的博莱霉素- 成型步骤。这项研究将利用酶学专业知识在 皮S群与耶尔逊细菌群的遗传、微生物学和 共同研究员S小组的发病机制专家破译了 毒力决定铁载体生物发生的分子机制。

Iron chelating siderophores and several peptide antibiotics are assembled by nonribosomal peptide synthase complexes via a thioltemplate mechanism. The siderophore Yersiniabactin, a key virulence factor in Yersinia infections, has both an aryl-N-Cap initiating biosynthesis and five membered sulfur heterocycles (thiazolines, thiazolidines resulting from cyclization of cysteines) that coordinate iron. The research proposed here involves determination of the genes responsible for yersiniabactin biosynthesis and characterization of the first enzymatic steps that involve activation of salicylic acid, amide bond formation to cysteine residues and cyclization to the thiazoline. We propose to purify YbtE and domains for the 200KD high molecular weight protein 2 of Y. pestis and analyze the anticipated enzymatic activity, including posttranslational phosphopantetheinylation (Ppant), salicyl-S-Ppant enzyme loading, salicyl-cys-S-enzyme formation, and cyclization to a salicyl-thiazoline-S-enzyme intermediate. These studies should decipher the logic of enzymatic assembly not only of virulence-conferring siderophores yersiniabactin and anguibactin but also for peptide antibiotics such as actinomycin, pristinamycin, and the antitumor agent bleomycin which use aryl N-cap imitation and/or thiazoline, oxazline- forming steps. The research will utilize the enzymology expertise in the PI s group and the yersiniabacteria genetic, microbiology and pathogenesis expertise in the co-investigator s group to decipher the molecular mechanisms for virulence determining siderophore biogenesis.