Oxygen Activation and Radical Transfer in Ribonucleotide Reductase from Pathogens

病原体核糖核苷酸还原酶的氧活化和自由基转移

• 批准号: 7526607
• 负责人: JOSEPH M BOLLINGER
• 金额: $ 46.4万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526607
• 关键词: Amino Acids; Antibiotics; Architecture; Aromatic Amino Acids; Binding; Biology; Catalysis; Chemicals; Chlamydia; Chlamydia trachomatis; Chlamydophila pneumoniae; Class; Complex; Computing Methodologies; Crystallography; Cysteine; DNA biosynthesis; DNA chemical synthesis; Deoxyribonucleotides; Development; Disease; Drug Delivery Systems; Electron Transport; Electrons; Enzymes; Escherichia coli; Event; Evolution; Free Radicals; Holoenzymes; Homo sapiens; Human; Hydrogen; Hydroxylamine; Immune Targeting; Immune response; Iron; Kinetics; Manganese; Mediating; Metals; Mycobacterium tuberculosis; Natural regeneration; Nitrogen; Nucleotides; Organism; Oxidation-Reduction; Oxygen; Pathway interactions; Pharmaceutical Preparations; Phenylalanine; Positioning Attribute; Process; Proteins; Protons; Public Health; Reaction; Reporting; Research; Resistance; Ribonucleotide Reductase; Ribonucleotide Reductase Inhibitor; Role; Simplexvirus; Site; Spectrum Analysis; Standards of Weights and Measures; Structure; System; Testing; Thermodynamics; Time; Tyrosine; Variant; Viral Cancer; Virus Diseases; abstracting; carboxylate; cofactor; design; hydroxyurea; novel; nucleoside diphosphate; oxidation; pathogen; repaired; three dimensional structure

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) provide deoxyribonucleotides for DNA synthesis and repair. The enzymes employ a conserved free-radical mechanism. Class I RNRs, including the human and Herpes Simplex Virus I enzymes, use a stable tyrosyl radical to initiate this mechanism and are validated drug targets. Several of the drugs function (at least in part) by reducing the tyrosyl radical. The tyrosyl radical is introduced into the enzyme by reaction of a di-iron(II) center with O2. Class I RNRs found in important human pathogens such as Chlamydia trachomatis and Mycobacterium tuberculosis lack the tyrosyl radical. The C. trachomatis RNR is, nevertheless, active. We recently showed that the C. trachomatis RNR uses a stable Mn(IV)/Fe(III) cofactor in place of the tyrosyl radical to initiates its reaction. The cofactor undergoes reduction to the Mn(III)/Fe(III) form to generate a protein radical that abstracts a hydrogen atom from the substrate. The Ct RNR is the first example of a manganese-dependent RNR, and its cofactor is the first example of a Mn/Fe redox center in biology. The cofactor is introduced, analogously to the tyrosyl radical in the conventional class I RNRs, by reaction of the reduced [Mn(II)/Fe(II)] metal center with O2. In this reaction, a Mn(IV)/Fe(IV) accumulates to a high level. In this project, we will elucidate the mechanisms of the formation and catalytic function of this novel cofactor. We will define the structures of its Mn(II)/Fe(II), Mn(IIII)/Fe(III), Mn(IV)/Fe(III) and Mn(IV)/Fe(IV) states by spectroscopic and computational methods and x-ray crystallography. We will understand how the protein protects the oxidized cofactor from adventitious reduction but then allows it to be reduced at the appropriate time to form the hydrogen-abstracting protein radical. We will study its chemical reactivity to uncover unique vulnerabilities that might be exploited in design of new drugs against the pathogens that use this type of RNR. Finally, we will compare the structures of closely related pairs of RNRs, of which one uses the standard tyrosyl radical and the other the novel Mn(IV)/Fe(III) cofactor, for clues to the design of both systems and the evolution of one from another. We will then attempt to use these clues to rationally convert one type of RNR into the other by changing crucial amino acids. PUBLIC HEALTH RELEVANCE: The enzyme ribonucleotide reductase (RNR) catalyzes the key step in DNA biosynthesis of all organisms and is a validated target for treatment of cancer and viral diseases. We recently reported that the class Ic RNR from the human pathogen Chlamydia trachomatis uses a novel redox cofactor (a heterobinuclear Mn/Fe cluster) to initiate catalysis. The structure and mechanism of this novel RNR will be elucidated to facilitate the rational development of class Ic RNR inhibitors that could be used to treat diseases caused by C. trachomatis and several other human pathogens (e.g. Chlamydia pneumoniae and Mycobacterium tuberculosis).

描述（由申请人提供）：核糖核苷酸还原酶（RNR）为DNA合成和修复提供脱氧核糖核苷酸。这些酶采用保守的自由基机制。I类RNR，包括人类和单纯疱疹病毒I酶，使用稳定的酪氨酰自由基来启动这种机制，并且是经验证的药物靶标。几种药物的功能（至少部分）是通过减少酪氨酰基自由基。酪氨酰基自由基通过二铁（II）中心与O2的反应引入酶中。在重要的人类病原体如沙眼衣原体和结核分枝杆菌中发现的I类RNR缺乏酪氨酰自由基。梭然而，沙眼RNR是活跃的。我们最近发现，C。沙眼RNR使用稳定的Mn（IV）/Fe（III）辅因子代替酪氨酰基自由基来引发其反应。辅因子经历还原成Mn（III）/Fe（III）形式以产生从底物提取氢原子的蛋白质自由基。Ct RNR是锰依赖性RNR的第一个例子，其辅因子是生物学中Mn/Fe氧化还原中心的第一个例子。类似于常规I类RNR中的酪氨酰基自由基，通过还原的[Mn（II）/Fe（II）]金属中心与O2的反应引入辅因子。在该反应中，Mn（IV）/Fe（IV）累积到高水平。在这个项目中，我们将阐明这种新的辅因子的形成和催化功能的机制。我们将通过光谱和计算方法以及X射线晶体学来定义其Mn（II）/Fe（II）、Mn（IIII）/Fe（III）、Mn（IV）/Fe（III）和Mn（IV）/Fe（IV）状态的结构。我们将了解蛋白质如何保护氧化辅因子免受偶然还原，但随后允许其在适当的时间被还原以形成夺氢蛋白质自由基。我们将研究它的化学反应性，以发现独特的漏洞，这些漏洞可能被用于设计针对使用这种RNR的病原体的新药。最后，我们将比较密切相关的RNR对的结构，其中一个使用标准酪氨酰自由基，另一个使用新型Mn（IV）/Fe（III）辅因子，以寻找这两个系统的设计和相互进化的线索。另一个。然后，我们将尝试利用这些线索，通过改变关键氨基酸，合理地将一种类型的RNR转化为另一种类型。公共卫生关系：核糖核苷酸还原酶（RNR）催化所有生物体DNA生物合成的关键步骤，是治疗癌症和病毒性疾病的有效靶点。我们最近报道，从人类病原体沙眼衣原体的Ic类RNR使用一种新的氧化还原辅因子（异双核锰/铁簇）启动催化。本文将阐明这一新的RNR的结构和作用机制，为合理开发用于治疗C.沙眼和其他几种人类病原体（如肺炎衣原体和结核分枝杆菌）。