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

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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).