ESR STUDY OF DIPHTHAMIDE BIOSYNTHESIS

敌草胺生物合成的ESR研究

• 批准号: 8364046
• 负责人: BORIS G DZIKOVSKI
• 金额: $ 0.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364046
• 关键词: Anabolism; Archaea; Binding; Biochemical; Biochemical Reaction; Electron Spin Resonance Spectroscopy; Enzymes; Funding; Genes; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Human; Iron; Methods; National Center for Research Resources; Peptide Elongation Factor 2; Play; Principal Investigator; Protein Biosynthesis; Proteins; Research; Research Infrastructure; Resources; Role; Shapes; Source; Spin Trapping; Sulfur; Technology; Tumor Suppressor Genes; United States National Institutes of Health; Work; Yeasts; adduct; cold temperature; cost; dimer; novel; oxidation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal or this project is to understand the enzymatic reaction mechanism of diphthamide biosynthesis. Diphthamide is a posttranslationally modified His residue in eukaryotic translation elongation factor 2 (EF-2) a GTPase required for the translocation step of ribosomal protein synthesis. One of the most intriguing facts about diphthamide is that one of the genes involved in its biosynthesis is found to be a tumor suppressor gene in human. Recently, Lin group provided structural and biochemical evidence showing that the first step of diphthamide biosynthesis in archaea uses a novel iron-sulfur cluster enzyme, DPH2 (Zhu and others 2009). ESR spectroscopy at 4- 15K in ACERT played a crucial role in identifying the [4Fe-4S] iron-sulfur cluster. The results obtained by different methods in Lin's group suggest that unlike known radical SAM enzymes, DPH2 does not form 5¿-deoxyadenosyl radicals. Instead, it breaks the other C-S bond of SAM and transfers the 3-amino-3-carboxylpropyl group to EF-2, possibly via a radical mechanism. To prove or disprove the proposed radical mechanism, we plan to spin-trap and identify the intermediates by the shape of ESR spectra of their adducts with DMPO and possibly other spin-traps. We recently detected some spin trapping adducts and work on their identification. Also, our next step will be a study of other proteins involved in the biosynthesis of diphthamide: yeast DPH1-DPH2 dimer, and yeast DPH3, DPH4. We expect that all these proteins have a Fe-S cluster bound. To identify the cluster, its spin and oxidation state we will record low temperature EPR at a wide field range. It will allow us to detect higher spin states (S= 3/2, 5/2, 7/2) or mixtures thereof.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 目标或该项目是了解二烷酰胺生物合成的酶促反应机制。 Diphthamide是一种在真核翻译伸长因子2（EF-2）的核糖体蛋白质合成步骤中所需的GTPase的跨化修饰。关于Diphthamide的最有趣的事实之一是，发现其生物合成的基因之一被发现是人类中的肿瘤抑制基因。最近，Lin组提供了结构和生化证据，表明古细菌中二乙酰胺生物合成的第一步使用了一种新型的铁硫簇DPH2（Zhu等，2009）。 ACERT 4-15K处的ESR光谱学在识别[4FE-4S]铁硫簇中起着至关重要的作用。 Lin组中不同方法获得的结果表明，与已知的自由基SAM酶不同，DPH2不形成5？ - 脱氧丁糖基自由基。取而代之的是，它破坏了SAM的另一个C-S键，并将3-氨基-3-羧基丙基转移到EF-2，这可能是通过自由基机制传递给EF-2。为了证明或反驳所提出的自由基机制，我们计划通过与DMPO和可能的其他自旋陷阱的加合物的ESR光谱形状旋转陷阱并识别中间体。我们最近检测到了一些自旋诱捕加合物，并在其识别方面工作。此外，我们的下一步将是对二乙酰胺生物合成的其他蛋白质的研究：酵母DPH1-DPH2二聚体和酵母DPH3，DPH4。我们希望所有这些蛋白质都具有Fe-S簇结合。为了识别簇，其自旋和氧化状态，我们将在广泛的场范围内记录低温EPR。它将允许我们检测到较高的自旋状态（s = 3/2、5/2、7/2）或混合。