MElll2_Y_Me_Fe_Mn_Cluster Assembly and Maintenance in Ribonucleotide Reductase

描述(申请人提供)：在所有提供DNA复制和修复所需单体前体的生物体中，核糖核苷酸还原酶催化核苷酸转化为脱氧核苷酸。Ia类和Ib类RNRs由两个亚基(？然后呢？)它们形成了活性(？2)n？2络合物。？2含有必需的双核-酪氨酸基(Y)辅因子，在来自大肠杆菌、酿酒酵母和人类的Ia类RNRs中是一个二铁簇(FeIII2-Y)，在来自大肠杆菌、枯草杆菌、血链球菌和许多病原微生物的Ib类RNRs中可能是一个二锰簇(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药物羟基脲还原，MeIII2-簇如何重新转化为活性辅因子。FeIII2-Y在Ia RNRs中的体外自组装研究表明，需要减少当量，并控制金属和氧化剂的输送。我们最近在大肠杆菌中发现了一种2Fe2S簇铁氧还蛋白(YfaE)，它在簇的生物合成和维持中发挥着电子转移的作用，并可能参与簇的形成 金属化。在酿酒酵母真核Ia簇的组装中，Dre2和Tah18被认为在生物合成和维持的电子传递中起着相似的作用，而Grx3/Grx4和Dre2/Tah18被认为在金属化中起到了类似的作用。虽然Ib类RNRs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，体内活性的辅因子是MnIII2-Y簇，其体外和体内组装需要一种不寻常的黄毒素NrdI。目前的建议集中于使用我们在过去几年开发的工具(全细胞EPR、抗体、活性分析、带有基因缺失的同源菌株和条件表达以及X射线结晶学)来获得体内蛋白质因子(E.Coli和S.cerevisiae)作用的证据。我们还使用时间分辨生物物理方法(停流、快速冷冻猝灭EPR和EXAFS光谱)研究了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和它们的宿主之间竞争控制金属作用的重要性，并为抗菌治疗提供了新的靶点。

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.