Publications

• Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase.

  Chen J.Y., Bodley J.W.

  The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the p ... >> More

  The inactivation of elongation factor 2 (EF-2) by diphtheria toxin requires the presence of a post-translationally modified histidine residue in EF-2. This residue, diphthamide, has the structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine. The present work was undertaken to study the pathway of diphthamide biosynthesis using diphtheria toxin-resistant yeast mutants (Chen. J.-Y., Bodley, J. W., and Livingston, D. M. (1985) Mol. Cell. Biol. 5, 3357-3360) which are defective in diphthamide formation. We demonstrate here that one of these mutants (dph5) contains a toxin-resistant form of EF-2 which can be converted in vitro to a toxin-sensitive form through the action of an enzyme present in other yeast strains. Both this toxin-resistant EF-2 and its modifying enzyme have been partially purified and evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase. In vitro complementation to diphtheria toxin sensitivity required S-adenosylmethionine, and when partially purified components were incubated with [methyl-3H]S-adenosylmethionine, label was incorporated specifically into EF-2. Hydrolysis of labeled EF-2 yielded diphthine (the unamidated form of diphthamide) and a single chromatographically separable labeling intermediate. We conclude that the S-adenosylmethionine:EF-2 methyltransferase adds at least the last two of the three methyl groups present in diphthine and that this modification is sufficient to create diphtheria toxin sensitivity. Evidence is also presented for the existence of an ATP-dependent amidating enzyme which catalyzes the final step in the biosynthesis of diphthamide in EF-2. << Less

  J Biol Chem 263:11692-11696(1988) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Dph7 catalyzes a previously unknown demethylation step in diphthamide biosynthesis.

  Lin Z., Su X., Chen W., Ci B., Zhang S., Lin H.

  Present on archaeal and eukaryotic translation elongation factor 2, diphthamide represents one of the most intriguing post-translational modifications on proteins. The biosynthesis of diphthamide was proposed to occur in three steps requiring seven proteins, Dph1-7, in eukaryotes. The functional a ... >> More

  Present on archaeal and eukaryotic translation elongation factor 2, diphthamide represents one of the most intriguing post-translational modifications on proteins. The biosynthesis of diphthamide was proposed to occur in three steps requiring seven proteins, Dph1-7, in eukaryotes. The functional assignments of Dph1-5 in the first and second step have been well established. Recent studies suggest that Dph6 (yeast YLR143W or human ATPBD4) and Dph7 (yeast YBR246W or human WDR85) are involved in the last amidation step, with Dph6 being the actual diphthamide synthetase catalyzing the ATP-dependent amidation reaction. However, the exact molecular role of Dph7 is unclear. Here we demonstrate that Dph7 is an enzyme catalyzing a previously unknown step in the diphthamide biosynthesis pathway. This step is between the Dph5- and Dph6-catalyzed reactions. We demonstrate that the Dph5-catalyzed reaction generates methylated diphthine, a previously overlooked intermediate, and Dph7 is a methylesterase that hydrolyzes methylated diphthine to produce diphthine and allows the Dph6-catalyzed amidation reaction to occur. Thus, our study characterizes the molecular function of Dph7 for the first time and provides a revised diphthamide biosynthesis pathway. << Less

  J. Am. Chem. Soc. 136:6179-6182(2014) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 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.

• The post-translational trimethylation of diphthamide studied in vitro.

  Moehring J.M., Moehring T.J.

  The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation ... >> More

  The amino acid diphthamide is a complex post-translational derivative of histidine that exists in eukaryotic and Archaebacterial elongation factor 2 (EF-2). Diphtheria toxin and Pseudomonas exotoxin A catalyze the transfer of an ADP-ribose residue from NAD to diphthamide, causing the inactivation of EF-2. We have used cytosolic extracts of mutant CHO-K1 cells to study the biosynthesis of diphthamide in vitro. We have identified chromatographically a precursor form of diphthamide that exists in one complementation group of mutant cells and have documented the addition of 3 methyl residues from S-adenosylmethionine to this precursor. We have identified the presence of methyltransferase capable of carrying out this reaction in vitro in cells of 15 diverse eukaryotic species. << Less

  J Biol Chem 263:3840-3844(1988) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.