ESR STUDY OF DIPHTHAMIDE BIOSYNTHESIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本项目的目的是了解生物合成联苯二酰胺的酶促反应机理。邻苯二甲酰胺（Diphthamide）是真核生物翻译延伸因子2（EF-2）中的一个后修饰的His残基，EF-2是核糖体蛋白合成的易位步骤所需的GT3。关于邻苯二甲酰胺最令人感兴趣的事实之一是，在人类中发现参与其生物合成的基因之一是肿瘤抑制基因。 最近，Lin Group提供了结构和生物化学证据，表明古细菌中的二苯二甲酰胺生物合成的第一步使用了一种新的铁硫簇酶DPH 2（Zhu等人，2009）。ACERT中4- 15 K的ESR谱在鉴定[4Fe-4S]铁硫团簇中起到了至关重要的作用。 Lin的研究小组通过不同方法获得的结果表明，与已知的自由基SAM酶不同，DPH 2不形成5-脱氧腺苷自由基。相反，它破坏SAM的另一个C-S键，并将3-氨基-3-羧基丙基转移到EF-2上，可能是通过自由基机制。为了证明或反驳所提出的自由基机制，我们计划自旋陷阱，并确定其与DMPO和可能的其他自旋陷阱加合物的ESR谱的形状的中间体。我们最近发现了一些自旋捕获加合物，并对其进行了鉴定。 此外，我们的下一步将是研究其他蛋白质参与生物合成的联苯二酰胺：酵母DPH 1-DPH 2二聚体，和酵母DPH 3，DPH 4。我们预期所有这些蛋白质都具有Fe-S簇结合。为了识别团簇，它的自旋和氧化态，我们将在宽场范围内记录低温EPR。它将允许我们检测更高的自旋状态（S= 3/2，5/2，7/2）或其混合物。

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.