Publications

• Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis.

  Zhu X., Dzikovski B., Su X., Torelli A.T., Zhang Y., Ealick S.E., Freed J.H., Lin H.

  Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the firs ... >> More

  Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer. << Less

  Mol. Biosyst. 7:74-81(2011) [PubMed] [EuropePMC]

• Identification of the proteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2.

  Liu S., Milne G.T., Kuremsky J.G., Fink G.R., Leppla S.H.

  Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 we ... >> More

  Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes. << Less

  Mol. Cell. Biol. 24:9487-9497(2004) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme.

  Zhang Y., Zhu X., Torelli A.T., Lee M., Dzikovski B., Koralewski R.M., Wang E., Freed J., Krebs C., Ealick S.E., Lin H.

  Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bon ... >> More

  Archaeal and eukaryotic translation elongation factor 2 contain a unique post-translationally modified histidine residue called diphthamide, which is the target of diphtheria toxin. The biosynthesis of diphthamide was proposed to involve three steps, with the first being the formation of a C-C bond between the histidine residue and the 3-amino-3-carboxypropyl group of S-adenosyl-l-methionine (SAM). However, further details of the biosynthesis remain unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron-sulphur-cluster enzyme, Dph2. Dph2 is a homodimer and each of its monomers can bind a [4Fe-4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5'-deoxyadenosyl radical. Instead, it breaks the C(gamma,Met)-S bond of SAM and generates a 3-amino-3-carboxypropyl radical. Our results suggest that P. horikoshii Dph2 represents a previously unknown, SAM-dependent, [4Fe-4S]-containing enzyme that catalyses unprecedented chemistry. << Less

  Nature 465:891-896(2010) [PubMed] [EuropePMC]