MOLECULAR ANALYSIS OF YERSINIABACTIN BIOSYNTHESIS

铁螯合铁载体和几种肽类抗生素， 由非核糖体肽合酶复合物通过巯基模板组装 机制 铁载体耶尔森氏杆菌素，一个关键的毒力因子， 耶尔森氏菌感染，具有芳基-N-帽起始生物合成和 五元硫杂环（噻唑啉，噻唑烷， 来自半胱氨酸的环化），其与铁配位。 研究 这里提出的方法涉及确定负责 耶尔森氏杆菌素的生物合成和第一酶的表征 涉及水杨酸活化、酰胺键形成 转化为半胱氨酸残基并环化为噻唑啉。 我们建议 纯化200 KD高分子量蛋白2的YbtE和结构域 鼠疫pestis并分析预期的酶活性，包括 翻译后磷酸泛酰巯基乙胺化（Ppant），水杨酰-S-Ppant 酶加载、水杨基-半胱氨酸-S-酶形成和环化成 水杨基-噻唑啉-S-酶中间体。 这些研究应该可以解释 酶促组装的逻辑不仅是赋予生物活性， 铁载体耶尔森菌素和anguibactin，但也用于肽 抗生素如放线菌素、普那霉素和抗肿瘤剂 博来霉素使用芳基N-帽模拟和/或噻唑啉、恶唑啉- 成型步骤 这项研究将利用酶学专业知识， PI组和耶尔森氏菌遗传学、微生物学和 共同研究者小组的发病机理专家， 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.