The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis

Min Dong, Emily E. Dando, Ilana Kotliar, Xiaoyang Su, Boris Dzikovski, Jack H. Freed, Hening Lin

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)777-782
Number of pages6
JournalJournal of Biological Inorganic Chemistry
Issue number6
StatePublished - Sep 1 2019

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Inorganic Chemistry


  • Diphthamide biosynthesis
  • Iron–sulfur cluster
  • Radical SAM enzyme

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