ESR STUDY OF DIPHTHAMIDE BIOSYNTHESIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本项目的目的是了解敌草胺生物合成的酶促反应机理。敌草胺是真核生物翻译延伸因子2(EF-2)中的一种翻译后修饰的His残基，是核糖体蛋白质合成转位步骤所必需的GTP酶。关于敌草胺最耐人寻味的事实之一是，在人类中发现了参与其生物合成的基因之一是肿瘤抑制基因。最近，LIN课题组提供的结构和生化证据表明，古生菌生物合成敌草胺的第一步使用了一种新的铁-硫簇酶DPH2(朱等人，2009)。在Acert中，4-15K的ESR谱在确定[4Fe-4S]铁硫团簇中起着至关重要的作用。 林的小组用不同方法得到的结果表明，与已知的自由基SAM酶不同，DPH2不形成5？-脱氧腺苷自由基。相反，它断裂了SAM的另一个C-S键，并可能通过自由基机制将3-氨基-3-羧丙基转移到EF-2上。为了证明或反驳所提出的自由基机制，我们计划通过中间体与DMPO的加合物的ESR谱形状以及可能的其他自旋陷阱来鉴定中间体。我们最近检测到了一些自旋捕获加合物，并对它们进行了鉴定。此外，我们的下一步将是研究参与双苯二甲胺生物合成的其他蛋白质：酵母DPH1-DPH2二聚体，以及酵母DPH3，DPH4。我们推测所有这些蛋白质都有一个Fe-S簇结合。为了确定团簇及其自旋和氧化态，我们将在宽场范围内记录低温EPR。这将使我们能够探测到更高的自旋态(S=3/2，5/2，7/2)或它们的混合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.