Iron-Sulfur Center Regulation and Crosstalk of two Radical SAM Modifiers by one Electron Transfer Protein in Yeast?

自由基SAM酶中的铁硫（FeS）中心在许多对所有细胞（包括我们自己的细胞）都至关重要的生化反应中使用电子进行还原性SAM切割和自由基形成。因此，自由基SAM酶的缺陷可引起人类疾病。例如，卵巢癌与自由基SAM酶（Dph1-Dph2）的突变有关，该酶用于二甲氰胺修饰EF2，这是真核生物中必不可少的mRNA翻译因子，而细长子（tRNA反密码子修饰复合物，具有自由基SAM催化亚基（Elp3））的缺陷与严重的神经病变有关。为了确保EF2和tRNA正常发挥作用，这两种修饰剂应该在任何时候都是活跃的。然而，显示这些变化的数据却表明情况并非如此。有趣的是，这两种酶共享一种电子转移蛋白：Kti11（又名Dph3）。Dph3/Kti11向Dph1-Dph2提供电子，并与Kti13配合，参与电子流向Elp3。这表明两种自由基SAM酶通过电子流向（或从）它们的FeS中心进行调控。原则上，Kti13-Kti11/Dph3复合物可以将电子送入两种修饰途径或将其限制在拉长子（Elp3）上。虽然有证据支持这两种选择，但Kti13在双苯二胺途径中的相关性是没有意义的，而且两种模型的组合，其中Kti13支持电子流向两个自由基SAM酶，但对每个都有不同的贡献，还不能排除。因此，我们的目标是研究Kti13的确切作用及其引导电子流从Kti11/Dph3到Dph1-Dph2或Elp3复合物中的FeS中心的潜力。利用酵母作为模型真核生物，这将为正确调节自由基SAM催化和防止非生物电子流向随机的、潜在有害的受体提供新的见解。此外，由于tRNA和EF2修饰因子与mRNA翻译有关，因此，对进出其FeS中心的电子流模式的阐明可能有助于了解两种自由基SAM酶之间的功能串扰。为了实现这些目标，我们将回答以下问题：Kti13能否将电子从Kti11/Dph3引导到不同酶（Elp3和/或Dph1-Dph2）的FeS中心？Kti11/Dph3（和Kti13）功能突变的分离是否揭示了FeS中心的差异调控？-在通过Kti11/Dph3或Kti13翻译mRNA时，两种自由基SAM酶之间是否存在串音？随着钼辅助因子（MOCO）合成的s转移和与自由基SAM化学密切相关的长链依赖trna的硫代化，以及与人类健康状况相关的MOCO和修饰trna的不适当水平，我们的项目具有长期的生物医学意义。因此，它完全符合SPP1927的“铁硫生命”重点，并且与参与mRNA翻译的自由基SAM酶的功能和适当调节有关，mRNA翻译是所有细胞（包括我们自己的细胞）合成蛋白质的重要步骤。

Iron-sulfur (FeS) centers in radical SAM enzymes use electrons for reductive SAM cleavage and radical formation in numerous biochemical reactions that are crucial for all cells (including our own). Accordingly, defects in radical SAM enzymes can cause disease in humans. For instance, ovarian cancer is linked to mutations in a radical SAM enzyme (Dph1-Dph2) for diphthamide modification of EF2, an essential mRNA translation factor in eukaryotes, and defects in Elongator, a tRNA anticodon modifying complex with a radical SAM catalytic subunit (Elp3), are associated with severe neuropathies. To ensure proper EF2 and tRNA functioning, both modifiers ought to be active at all times. However, data showing the modifications do change, suggest otherwise. Intriguingly, both enzymes share one electron transfer protein: Kti11 (aka Dph3). Dph3/Kti11 donates electrons to Dph1-Dph2, and in complex with Kti13, is implicated in electron flow to Elp3. This suggests regulation of two radical SAM enzymes via electron flow to (and from) their FeS centers. In principle, the Kti13-Kti11/Dph3 complex may feed electrons into both modification pathways or restrict them to Elongator (Elp3). Although there is evidence to support either option, the relevance of Kti13 in the diphthamide pathway is moot, and a combination of both models, where Kti13 supports electron flow to both radical SAM enzymes but with a differential contribution to each, cannot be excluded yet.Hence, we aim to study the precise role of Kti13 and its potential to guide electron flow from Kti11/Dph3 to FeS centers in the Dph1-Dph2 or Elongator (Elp3) complexes. Using yeast as a model eukaryote, this will provide novel insights into proper regulation of radical SAM catalysis and prevention of non-biological electron flow to random, potentially harmful acceptors. Moreover, with the tRNA and EF2 modifiers being linked to mRNA translation, elucidation of the mode of electron flow to (and from) their FeS centers may inform about functional cross-talk among both radical SAM enzymes. To achieve these aims we will answer the following questions:- Can Kti13 guide electrons from Kti11/Dph3 to FeS centers in different enzymes (Elp3 and/or Dph1-Dph2)?- Do separation of Kti11/Dph3 (and Kti13) function mutations reveal differential FeS center regulation?- Is there cross-talk among both radical SAM enzymes in mRNA translation via Kti11/Dph3 or Kti13?With S-transfer for molybdenum cofactor (MOCO) synthesis and thiolation of Elongator dependent tRNAs intimately linked to radical SAM chemistry and inappropriate levels of MOCO and modified tRNAs related to human health conditions, our project is biomedically significant in the long term. So it neatly fits the SPP1927 focus Iron-Sulfur for Life and is relevant with regards to function and proper regulation of radical SAM enzymes involved in mRNA translation, an essential step in protein synthesis by all cells (including our own).