KatG Peroxidase in Isoniazid Activation and Resistance

KatG 过氧化物酶在异烟肼激活和耐药中的作用

• 批准号: 6434283
• 负责人: WHYTE G OWEN
• 金额: $ 17.92万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6434283
• 关键词: Mycobacterium tuberculosis; Raman spectrometry; X ray crystallography; bacterial genetics; bacterial proteins; binding sites; drug resistance; electron spin resonance spectroscopy; enzyme activity; enzyme structure; isoniazid; nuclear magnetic resonance spectroscopy; oxidation; peroxidases; point mutation; site directed mutagenesis; tuberculosis

DESCRIPTION (Adapted from the Applicant's Abstract): Tuberculosis is one of the leading causes of death worldwide from an infectious disease. Isoniazid is a front line antibiotic used in the treatment of tuberculosis. The Mycobacterium tuberculosis heme catalase/peroxidase KatG is responsible for activating isoniazid to a reactive biocidal species. Antibiotic resistance to isoniazid is a growing concern and can occur by deletions or point mutations in the katG gene. One of these, a serine-to-threonine substitution at position 315, KatG(S315T) is found in about 50 percent of clinical isolates resistant to isoniazid. Our research seeks to understand the mechanism of isoniazid activation and molecular basis for drug resistance caused by the S315T and other point mutations in KatG using biochemical, genetic, and spectroscopic techniques. KatG will be purified from recombinant and native sources and the activities and spectroscopic properties compared. Besides a catalase/peroxidase activity, KatG can also catalyze several other reactions including Mn2+ peroxidase, P450-like oxygenase, and peroxynitritase activities. Which of these or possibly other redox reactions are responsible for isoniazid oxidation and activation will be tested using wild-type and mutant KatG proteins to investigate the mechanism of activation. Genetic methods investigating the role of superoxide in isoniazid activation are proposed. Optical, EPR, NMR, and resonance Raman spectroscopies are revealing subtle differences in the heme active site of wild-type KatG and KatG(S315T). These spectroscopic techniques will be applied to various forms of wild-type and mutant enzymes in order to elucidate the molecular basis for reactivity toward isoniazid and the reduced rate of isoniazid oxidation by KatG(S315T). NMR relaxation measurements and x-ray crystallography will be used to map the isoniazid binding site on both enzymes to determine whether subtle differences in distance and/or orientation are responsible for the reduced turnover of drug by the mutant enzyme. Steady-state and rapid kinetic techniques will follow ligand-binding rates to the heme iron and the formation and decay of reactive intermediates in the catalytic cycle for both wild-type KatG and KatG(S3 1ST) to determine whether the S3 1ST mutation affects one of the steps in the catalytic cycle. EPR spectroscopy and spin-traps are being used to trap reactive intermediates. The stable products formed in this reaction are being characterized by mass spectrometry to reveal chemical information about the nature of intermediates in the reaction of isoniazid oxidation by KatG. Site directed mutagenesis of residues also implicated in isoniazid resistance other than S3 15 will be generated and the effects on enzyme activities and spectroscopic properties examined to determine structural information about the mutant enzymes. Analogs of isoniazid will be used as additional biochemical and spectroscopic probes of the reaction mechanism and molecular basis for drug resistance.

描述（根据申请人的摘要改编）：结核病是其中之一 全世界因传染病而导致死亡的主要原因。 Isoniazid是一个 前线抗生素用于治疗结核病。分枝杆菌 结核血红素过氧化氢酶/过氧化物酶KATG负责激活 异念珠菌对反应性杀菌物种。抗生素对异尼二氮的抗性为 越来越关注的问题，可以通过katg中的删除或点突变发生 基因。其中之一是位于315位的丝氨酸向硫酸盐的替代 在大约50％的临床分离株中发现了Katg（S315T） 异尼济。我们的研究旨在了解Isoniazid的机制 由S315T引起的耐药性的激活和分子基础 使用生化，遗传和光谱镜的KATG中的其他点突变 技术。 KATG将从重组和本地来源纯化，以及 比较活动和光谱特性。除了过氧化氢酶/过氧化物酶 活动，KATG还可以催化其他几种反应，包括MN2+ 过氧化物酶，P450样氧酶和过氧亚硝酸酶活性。其中哪一个 或其他氧化还原反应可能导致异念珠化氧化和 将使用野生型和突变型KATG蛋白对激活进行激活 研究激活的机理。研究角色的遗传方法 提出了在异念珠菌激活中的超氧化物的。光学，EPR，NMR和 共振拉曼光谱显示出血红素的细微差异 野生型Katg和Katg（S315T）的活跃部位。这些光谱技术 将应用于各种形式的野生型和突变酶，以便 阐明分子基础对异尼二氮齐的反应性，并降低 Katg（S315T）的异念珠菌氧化速率。 NMR放松测量和 X射线晶体学将用于绘制这两个Isoniazid结合位点 酶确定距离和/或方向的细微差异是否存在 负责通过突变酶减少药物营业额。 稳态和快速动力学技术将遵循配体结合率 血红素铁以及反应性中间体的形成和衰变 野生型Katg和Katg（S3 1）的催化循环，以确定是否是否 S3第一突变会影响催化循环中的步骤之一。 EPR 光谱和自旋陷阱被用于捕获反应性中间体。这 在此反应中形成的稳定产品的特征是质量 光谱法揭示了有关中间体性质的化学信息 在katg的异尼二嗪氧化反应中。站点定向诱变 除了S3 15以外的等异氮二氮的抗性，残留物还将 生成以及对酶活性和光谱特性的影响 检查以确定有关突变酶的结构信息。类似物 异念珠菌将用作附加的生化和光谱探针 耐药性的反应机制和分子基础。