Abstract
Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe–4S] cluster-containing radical SAM enzyme, Dph1–Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe–4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1–Dph2 heterodimer, the [4Fe–4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1–Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1–Dph2 heterodimer and may help to understand how the Fe–S clusters in radical SAM enzymes are reduced in biology.
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Acknowledgements
This work was supported by grants from the National Institutes of Health National Institute of General Medical Sciences GM088276 to H.L.
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Dong, M., Dando, E.E., Kotliar, I. et al. The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis. J Biol Inorg Chem 24, 777–782 (2019). https://doi.org/10.1007/s00775-019-01702-0
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DOI: https://doi.org/10.1007/s00775-019-01702-0