METALLOENZYME AND MICROCIN B17 SYNTHETASE

金属酶和小菌素 B17 合成酶

• 批准号: 6179813
• 负责人: DEBORAH B ZAMBLE
• 金额: $ 3.24万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6179813
• 关键词: antibacterial agents; atomic absorption spectrometry; chemical synthesis; cofactor; drug design /synthesis /production; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; mass spectrometry; metalloenzyme; oxazoles; site directed mutagenesis; stoichiometry; thiazoles; western blottings

The importance of unusual ring structures in a diverse array of therapeutic agents suggests that the ability to engineer compounds containing these moieties could lead to new, medically useful drugs. The ribosomally synthesized antibacterial agent, Microcin B17, is generated following post-translational modifications that produce both oxazole and thiazole rings in the polypeptide precursor. The enzyme responsible for this activity, Microcin B17 synthetase, provides an ideal system to study heterocycle formation because all of the genes for the multi-protein complex have been cloned and biochemical assays to monitor substrate modification have been established. This proposal describes studies that explore the mechanism of action of one enzymatic component, McbB, which may employ zinc as a cofactor. First, the stoichiometry of zinc ions bound by McbB and the metal requirements for catalytic activity will be established. The second set of experiments, which include several types of spectroscopy and site-directed mutagenesis, will examine the metal coordination site(s) and the influence of the metal ligands on substrate modification. Finally, McbB mutants, as well as modified substrates, will be used to elucidate the details of the enzymatic mechanism of oxazole and thiazole synthesis.

不同寻常的环结构在多种治疗剂中的重要性表明，设计含有这些部分的化合物的能力可能会产生新的、医学上有用的药物。核糖体合成的抗菌剂 Microcin B17 是在翻译后修饰后产生的，在多肽前体中产生恶唑和噻唑环。负责这种活性的酶 Microcin B17 合成酶为研究杂环形成提供了一个理想的系统，因为多蛋白复合物的所有基因都已被克隆，并且监测底物修饰的生化测定也已建立。该提案描述了探索一种酶成分 McbB 作用机制的研究，该成分可能采用锌作为辅助因子。首先，将确定 McbB 结合的锌离子的化学计量和催化活性的金属要求。第二组实验包括几种类型的光谱学和定点诱变，将检查金属配位位点以及金属配体对底物修饰的影响。最后，McbB 突变体以及修饰的底物将用于阐明恶唑和噻唑合成的酶促机制的细节。