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: 8983453
• 负责人: Elizabeth J Blaesi
• 金额: $ 5.24万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8983453
• 关键词: Aerobic Bacteria; Affect; Amino Acid Sequence; Bacteria; Base Composition; Binding; Biological Assay; Catalysis; Cell Nucleus; Chemicals; Chlamydia trachomatis; Coupled; 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; Methionine; Methods; 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; 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; killings; member; pathogen; protein complex; public health relevance; research study; theories; therapeutic target

 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（由申请人提供）：核糖核苷酸还原酶（RNR）通过还原相应的核糖核苷酸为DNA合成提供脱氧核苷酸前体，根据启动催化的辅因子分为三类。 I 类包含来自大肠杆菌 (Ec) 等需氧细菌和包括人类在内的高等真核生物的 RNR，进一步细分为三个亚类，部分通过双金属辅助因子（Fe2、Mn2 和 MnFe）的特性进行区分对于子类 a、b 和 c，分别）在  亚基中直接或间接生成保守的半胱氨酸自由基a 亚基中的 (Cys•) 引发剂已经确定，对于原型 Ia Ec RNR，Cys• 的生成是通过构象门控、长距离质子耦合电子转移 (PCET) 机制进行的，并且该步骤的重要细节已确定。本文的研究计划旨在以同等详细程度阐明 Ib 和 Ic RNR 中的 PCET 机制，这一目标将通过允许的捕获策略来实现。通过穆斯堡尔、电子顺磁共振和磁圆二色光谱技术将 PCET 产物状态与静止（反应物）状态进行比较。此外，我们的初步证据表明存在先前未被识别的第四子类（Id 类），其中。由于甚至缺乏对 Id 类 RNR 的基本了解，因此辅因子的组成、结构和 Cys• 生成机制仍有待确定。测定将针对该酶类进行优化，并与单周转实验和辅因子组成的光谱分析结合使用，以鉴定功能性辅因子和活性辅因子的产物状态，并将通过晶体学和光谱分析来确定其结构和状态。在这里，采用定点诱变、化学还原剂和/或基于机制的灭活剂的捕获策略将能够制备 PCET 产物状态，以鉴定功能。辅因子并定义其结构。