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

• 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
• 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

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？）它们形成了活性（？2）n？2复杂。? 2含有必需的双核酪氨酰基自由基（Y）辅因子，其在来自E. coli、S.酿酒酵母和人类是二铁簇（FeIII 2-Y），而在Ib类RNR中来自E. coli，B.枯草芽孢杆菌S.可能是一个二锰簇（MnIII 2- 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Y 在细胞内被内源性还原剂或化疗药羟基脲还原，MeIII 2-簇如何再转化为活性辅因子。FeIII 2-Y自组装研究 在体外的Ia RNR中，已经证明需要还原当量以及受控的金属和氧化剂递送。我们最近在E.大肠杆菌中，2Fe 2S簇铁氧还蛋白（YfaE）在簇生物合成和维持中起电子传递作用，可能参与簇形成 金属化。在真核细胞Ia类簇的组装中，S.提出了酿酒酵母、Dre 2和Tah 18在用于生物合成和维持的电子转移中起类似的作用，并且提出了Grx 3/Grx 4和Dre 2/Tah 18在金属化中起作用。而Ib类RNR也可以自组装FeIII 2-Y 我们最近的研究表明，MnIII 2-Y是一种在核苷酸还原中具有活性的辅因子 成簇及其在体外和体内组装需要一种不寻常黄素氧还蛋白NrdI。目前的建议集中在获得蛋白质因子在体内作用的证据（E。coli和革兰氏阳性菌S.酿酒酵母），使用我们在过去几年中开发的工具（全细胞EPR、抗体、活性测定、具有基因缺失和条件性表达的等基因菌株以及X射线晶体学）。我们也在研究MnIII 2-Y的作用机制 使用时间分辨生物物理方法（停止流动、快速冷冻淬灭EPR和EXAFS光谱）的辅因子装配。对很久以前假设的但难以捉摸的Mn-RNR的观察提高了病原体和其宿主之间竞争受控金属化的重要性，并为抗菌治疗提出了新的靶点。