MElll2_Y_Me_Fe_Mn_Cluster Assembly and Maintenance in Ribonucleotide Reductase

核糖核苷酸还原酶中的MEll12_Y_Me_Fe_Mn_簇组装和维护

基本信息

批准号：
8434673
负责人：
JOANNE STUBBE
金额：
$ 53.44万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-02 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8434673
关键词：
Anabolism Anti-Bacterial Agents Antibodies Architecture Binding Biological Assay Cells Complex Cysteine DNA Repair DNA biosynthesis Diphosphates Electron Nuclear Double Resonance Electron Transport Ensure Escherichia coli Ferredoxin Flavodoxin Free Radicals Freezing Gene Deletion Growth Heme Housing Human Hydrogen In Vitro Iron Ligation Maintenance Mediating Metals Methods Molecular Chaperones Nucleosides Nucleotides Organism Oxidants Pathway interactions Peptides Phylogenetic Analysis Play Proteins Reaction Reducing Agents Ribonucleotide Reductase Role Saccharomyces cerevisiae Spectrum Analysis Therapeutic Therapeutic Intervention Therapeutic Uses X-Ray Crystallography Yeasts antitumor agent cofactor electron donor hydroxyurea in vivo interest microorganism pathogen public health relevance repaired self assembly small molecule therapeutic target time use tool

项目摘要

DESCRIPTION (provided by applicant): Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides in all organisms supplying the monomeric precursors required for DNA replication and repair. The class Ia and Ib RNRs are composed of two subunits (? and ?) which form the active (?2)n?2 complex. ?2 houses the essential dinuclear-tyrosyl radical (Y) cofactor which in the class Ia RNRs from E. coli, S. cerevisiae, and humans is a diferric cluster (FeIII2-Y), and in the class Ib RNRs from E. coli, B. subtilis, S. sanguinis and many pathogenic microorganisms is likely, a dimanganese cluster (MnIII2- Y). We are interested in how these cofactors are biosynthesized and repaired, i.e., if the Y is reduced inside the cell by endogenous reductants or the chemotherapeutic hydroxyurea, how the MeIII2-cluster is re-converted to active cofactor. Studies of self-assembly of FeIII2-Y in the Ia RNRs in vitro have demonstrated the requirement for reducing equivalents, and controlled metal and oxidant delivery. We have recently discovered in E. coli, a 2Fe2S cluster ferredoxin (YfaE) that plays a role in electron transfer in cluster biosynthesis and maintenance and likely is involved in cluster metallation. In assembly of the eukaryotic class Ia cluster in S. cerevisiae, Dre2 and Tah18 are proposed to play a similar role in electron transfer for biosynthesis and maintenance and Grx3/Grx4 and Dre2/Tah18 are proposed to play a role in metallation. While the class Ib RNRs can also self-assemble FeIII2-Y active in nucleotide reduction, our recent studies suggest that the active cofactor in vivo is MnIII2-Y cluster and that its assembly in vitro and in vivo require an unusual flavodoxin, NrdI. The present proposal is focused on obtaining evidence for the roles of the protein factors in vivo (E. coli and S. cerevisiae) using the tools we have developed over the last few years (whole cell EPR, antibodies, activity assays, isogenic strains with gene deletions and conditional expression and X-ray crystallography). We are also investigating the mechanism of MnIII2-Y cofactor assembly using time resolved biophysical methods (stopped flow, rapid freeze quench EPR and EXAFS spectroscopies). The observation of the long ago postulated, but elusive, Mn-RNR raises the importance of the competition between pathogens and their hosts for controlled metallation and suggests new targets for antibacterial therapeutics.

描述（由申请人提供）：核糖核苷酸还原酶催化所有生物体中核苷酸向脱氧核苷酸的转化，提供DNA复制和修复所需的单体前体。 Ia 类和 Ib 类 RNR 由两个亚基 (? 和 ?) 组成，形成活性 (?2)n?2 复合物。 ?2 包含必需的双核酪氨酰自由基 (Y ) 辅助因子，其在来自大肠杆菌、酿酒酵母和人类的 Ia 类 RNR 中是一个二铁簇 (FeIII2-Y )，而在来自大肠杆菌的 Ib 类 RNR 中是一个二铁簇 (FeIII2-Y )大肠杆菌、枯草芽孢杆菌、血链球菌和许多病原微生物可能是二锰簇 (MnIII2-Y)。我们感兴趣的是这些辅因子是如何生物合成和修复的，即，如果细胞内的 Y 被内源性还原剂或化疗羟基脲还原，MeIII2 簇如何重新转化为活性辅因子。 Ia RNR 中 FeIII2-Y 体外自组装的研究表明需要还原当量以及受控金属和氧化剂输送。我们最近在大肠杆菌中发现了一种 2Fe2S 簇铁氧还蛋白 (YfaE)，它在簇生物合成和维持中的电子转移中发挥作用，并且可能参与簇金属化。在酿酒酵母真核 Ia 类簇的组装中，Dre2 和 Tah18 被认为在生物合成和维持的电子转移中发挥类似的作用，Grx3/Grx4 和 Dre2/Tah18 被认为在金属化中发挥作用。虽然 Ib 类 RNR 也可以自组装具有核苷酸还原活性的 FeIII2-Y，但我们最近的研究表明，体内的活性辅因子是 MnIII2-Y 簇，并且其体外和体内组装需要一种不寻常的黄素氧还蛋白 NrdI 。本提案的重点是使用我们在过去几年开发的工具（全细胞 EPR、抗体、活性测定、同基因菌株与基因缺失和条件表达以及 X 射线晶体学）。我们还在利用时间分辨生物物理方法（停流、快速冷冻淬灭 EPR 和 EXAFS 光谱）研究 MnIII2-Y 辅因子组装的机制。对很久以前假设但难以捉摸的 Mn-RNR 的观察提高了病原体与其宿主之间对受控金属化的竞争的重要性，并为抗菌治疗提出了新的靶点。