Mechanisms of Radical Translocation in Class I Ribonucleotide Reductases

I 类核糖核苷酸还原酶中自由基易位的机制

• 批准号: 9318548
• 负责人: Elizabeth J Blaesi
• 金额: $ 5.08万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9318548
• 关键词: Aerobic Bacteria; Affect; Amino Acid Sequence; Anaerobic Bacteria; Bacteria; Biological Assay; Catalysis; Cell Nucleus; Chemicals; Chlamydia trachomatis; Coupled; Crystallization; Cysteine; DNA biosynthesis; Data; Deoxyribonucleotides; Electron Spin Resonance Spectroscopy; Electron Transport; Enzymes; Escherichia coli; Eukaryota; Experimental Designs; Family; Flavobacteria; Flavobacterium genus; Generations; Geometry; Goals; Hand; Human; Hydrogen; Investigation; Kinetics; Ligands; Mediating; Metals; Methods; Molecular Conformation; Mossbauer Spectroscopy; Mycobacterium tuberculosis; Nature; Organism; Oxidants; Oxidation-Reduction; Play; Preparation; Process; Proteins; Protons; Reducing Agents; Research; Rest; Ribonucleotide Reductase; Ribonucleotides; Ribose; Roentgen Rays; Role; S-Adenosylhomocysteine; Site-Directed Mutagenesis; Solvents; Spectrum Analysis; Staphylococcus aureus; Structure; Techniques; Testing; Work; absorption; antimicrobial drug; base; circular magnetic dichroism; cobamamide; cofactor; density; divalent metal; electron nuclear double resonance spectroscopy; electronic structure; experimental study; geometric structure; killings; member; pathogen; protein complex; public health relevance; targeted treatment; theories

 DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs), which provide the deoxynucleotide precursors for DNA synthesis by reducing the corresponding ribonucleotides, are divided into three classes on the basis of the cofactor that initiates catalysis. Historically, class I, which comprises the RNRs from aerobic bacteria such as Escherichia coli (Ec) and higher eukaryotes including humans, is further subdivided into three subclasses that are distinguished, in part, by the identity of the dimetal cofactor (Fe2, Mn2, and MnFe for subclasses a, b, and c, respectively) in the  subunit that either directly or indirectly generates a consered cysteine radical (Cys•) initiator in the a subunit. It has been established for the archetypal clss Ia Ec RNR that Cys• generation occurs by a conformationally gated, long distance proton-coupled electron transfer (PCET) mechanism, and important details of this step have been defined. The research plan herein aims to elucidate the PCET mechanisms in the Ib and Ic RNRs at an equivalent level of detail. This goal will be achieved through trapping strategies that permit the PCET product state to be compared to the resting (reactant) state by Mössbauer, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic techniques. In addition, our preliminary evidence suggests the existence of a previously unrecognized, fourth subclass (class Id), in which the cofactor's composition, structure, and mechanism of Cys• generation remain to be determined. As even a basic understanding of the class Id RNRs is lacking, multi-turnover kinetic assays will be optimized for this enzyme class, and used in conjunction with single-turnover experiments and spectroscopic analysis of cofactor composition to identify the functional cofactor. PCET reactant and product states of the active cofactor will b interrogated crystallographically and spectroscopically to define their structures and the mechanism of radical initiation. Here again, trapping strategies employing site-directed mutagenesis, chemical reductants, and/or mechanism-based inactivators will enable preparation of the PCET product state both to identify the functional cofactor and define its structure.

 描述（由适用提供）：核糖核苷酸还原酶（RNR），通过减少相应的核糖核苷酸来为DNA合成提供脱氧核苷酸前体，根据辅助因子的辅助因子分为三类，从而启动催化。从历史上看 I类包括有氧细菌（例如大肠杆菌（EC）和包括人类在内的较高真核生物）的RNR，进一步细分为三个子类，部分通过二胺辅因子（Fe2，MN2，MN2，MN2，MNF和MNFE）的属性分为cofactor（dimetal cofactor）的识别，分别是a和mnfe a subclass a a，b subs a a，b and b and b and b and b and b and b and b and cy and and and and and and and and c。在A亚基中生成一个被构成的半胱氨酸自由基（CYS•）引发剂。为cys的原型CLSS IA EC RNR建立•通过构象门控，长距离质子耦合电子传递（PCET）机制发生，并且已经定义了此步骤的重要细节。本文的研究计划旨在以等效的细节水平阐明IB和IC RNR中的PCET机制。将通过诱使PCET产物状态与Mössbauer，电子顺磁共振和磁性二分法光谱技术进行比较（反应物）状态的捕获策略来实现此目标。此外，我们的初步证据表明，存在先前未知的第四个子类（类ID），其中辅助因子的组成，结构和机制•生成仍有待确定。由于缺乏对类ID RNR的基本理解，该酶类的多转化动力学测定也将被优化，并与单转弯实验和辅因子组成的光谱分析一起使用，以识别功能性辅因子。活性辅助因子的PCET反应物和产物状态将在晶体学和光谱上进行询问，以定义其结构和自由基倡议的机制。再次，在这里，采用定向诱变，化学还原器和/或基于机制的灭活剂的捕获策略将使PCET产品状态能够识别功能性辅助因子并定义其结构。