Chen JY et al. (1988) J Biol Chem "Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase."

Bodley JW, Chen JY

[J Biol Chem, 1988]

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.

Dunlop PC et al. (1983) J Biol Chem "Biosynthetic labeling of diphthamide in Saccharomyces cerevisiae."

Dunlop PC, Bodley JW

[J Biol Chem, 1983]

Diphthamide, the post-translational amino acid derivative in the diphtheria toxin-modification site of protein synthesis elongation factor 2 (EF-2), has the proposed structure 2-[3-carboxyamido-3-(trimethylammonio)propyl]histidine (Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) J. Biol. Chem. 255, 10710-10716). The identification of the biosynthetic precursors of diphthamide would provide a means of evaluating its proposed structure and determining if the amino acid occurs in proteins other than EF-2. Toward this end, yeast were grown on potential radiolabeled precursors and the resulting radiolabeled protein was hydrolyzed in acid. The acid hydrolysates were subjected to amino acid analysis with a program optimized to resolve diphthine, the acid hydrolysis product of diphthamide, from the common amino acids. Radiolabel from [beta-3H]histidine, [alpha-3H]methionine, and [Me-3H] methionine was found to be incorporated into diphthine in a molar ratio of 1:1:3 while that of [35S]methionine was not incorporated. These results are in accord with the proposed structure of diphthamide and suggest that in its biosynthesis the backbone and 3 methyl groups of methionine are added to a histidine residue in the peptide chain of EF-2. These labeling experiments show that diphthine (diphthamide) constitutes approximately 6 X 10(-6) mol fraction of the total amino acids in yeast protein hydrolysates. Estimates of the amount of diphthamide present in the diphtheria toxin-modification site of EF-2 indicate that it constitutes from 4.5 to 9 X 10(-6) mol fraction of the total amino acids in yeast protein. The present evidence suggests that diphthamide occurs only in EF-2.

Chen JY et al. (1985) Mol Cell Biol "Diphtheria toxin-resistant mutants of Saccharomyces cerevisiae."

Livingston DM, Chen JY, Bodley JW

[Mol Cell Biol, 1985]

We developed a selection procedure based on the observation that diphtheria toxin kills spheroplasts of Saccharomyces cerevisiae (Murakami et al., Mol. Cell. Biol. 2:588-592, 1982); this procedure yielded mutants resistant to the in vitro action of the toxin. Spheroplasts of mutagenized S. cerevisiae were transformed in the presence of diphtheria toxin, and the transformed survivors were screened in vitro for toxin-resistant elongation factor 2. Thirty-one haploid ADP ribosylation-negative mutants comprising five complementation groups were obtained by this procedure. The mutants grew normally and were stable to prolonged storage. Heterozygous diploids produced by mating wild-type sensitive cells with the mutants revealed that in each case the resistant phenotype was recessive to the sensitive phenotype. Sporulation of these diploids yielded tetrads in which the resistant phenotype segregated as a single Mendelian character. From these observations, we concluded that these mutants are defective in the enzymatic steps responsible for the posttranslational modification of elongation factor 2 which is necessary for recognition by diphtheria toxin.