Oxygen Activation and Radical Transfer in Ribonucleotide Reductase from Pathogens

描述（由申请人提供）：核糖核苷酸还原酶（RNRs）提供用于DNA合成和修复的脱氧核糖核苷酸。酶采用保守的自由基机制。I类rrna，包括人类和单纯疱疹病毒I型酶，使用稳定的酪氨酸自由基启动这一机制，是经过验证的药物靶点。有几种药物（至少部分地）通过减少酪氨酸自由基起作用。酪氨酸自由基通过二铁（II）中心与氧的反应被引入酶中。在沙眼衣原体和结核分枝杆菌等重要的人类病原体中发现的I类rnr缺乏酪氨酸自由基。然而，沙眼衣原体RNR是活跃的。我们最近发现沙眼衣原体RNR使用稳定的Mn(IV)/Fe（III）辅助因子代替酪氨酸自由基来启动其反应。辅因子被还原成Mn(III)/Fe（III）的形式，生成从底物中提取氢原子的蛋白质自由基。Ct RNR是锰依赖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。辅助因子的引入，类似于传统的I类RNRs中的酪基自由基，是通过还原的[Mn(II)/Fe(II)]金属中心与O2反应而引入的。在这个反应中，Mn(IV)/Fe（IV）积累到很高的水平。在本项目中，我们将阐明这种新型辅因子的形成机制和催化功能。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）态的结构。我们将了解蛋白质是如何保护氧化辅因子免受不确定还原的，但随后允许它在适当的时候被还原，形成吸氢的蛋白质自由基。我们将研究它的化学反应性，以发现独特的弱点，这些弱点可能在设计针对使用这种类型RNR的病原体的新药时被利用。最后，我们将比较密切相关的RNRs对的结构，其中一个使用标准的酪氨酸自由基，另一个使用新的Mn(IV)/Fe（III）辅因子，为这两个系统的设计和彼此的进化提供线索。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RNR转化为另一种RNR。公共卫生相关性：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。我们最近报道了来自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助因子（异双核Mn/Fe簇）来启动催化。该新型RNR的结构和机制将被阐明，以促进Ic类RNR抑制剂的合理开发，这些抑制剂可用于治疗由沙眼衣原体和其他几种人类病原体（如肺炎衣原体和结核分枝杆菌）引起的疾病。

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).