DPH5 encodes the S-adenosylmethionine-dependent methyltransferase of the second step of diphthamide biosynthesis on translation elongation factor 2 (eEF2). It belongs to the tetrapyrrole (uroporphyrinogen III) methylase fold family and acts on the 2-(3-amino-3-carboxypropyl)histidine (ACP-His699) made by the Dph1-Dph2-Dph3 step. Yeast Dph5 transfers four methyl groups: three to the amino group (trimethylammonio) and one to the carboxylate, giving diphthine methyl ester, which Dph7 (RRT2) then hydrolyses to diphthine before Dph6 amidates it. This tetramethylation distinguishes the eukaryotic enzyme from archaeal Dph5, which makes N-trimethylated product without esterification. dph5 null mutants are viable, lack diphthamide, and are resistant to diphtheria toxin and sordarin. Dph5 is a soluble cytoplasmic protein.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004164 diphthine synthase activity | RCA GO_REF:0000123 | MODIFY | Summary: YeastPathways RCA (PWY-6482-1) assigns DPH5 the three single-methyl transfers ending at diphthine and maps them to diphthine synthase activity. Reason: GO:0004164 describes trimethylation of ACP-histidine to diphthine and is taxon-restricted to Archaea in GO. Yeast Dph5 also methylates the carboxylate and produces diphthine methyl ester (Lin et al. 2014), which is exactly GO:0141133, the Eukaryota-specific sister term already supported here by IDA/IMP. Proposed replacements: diphthine methyl ester synthase activity Supporting Evidence: PMID:24739148 Yeast Dph5 catalyzes the methylation of the amino and the carboxylate groups of ACP, generating methylated diphthine (4a). PMID:24739148 also reported that archaeal Dph5 catalyzes N-trimethylation of the ACP group PMID:24739148 yeast Dph5 catalyzes tetramethylation of the ACP group |
| GO:0005737 cytoplasm | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Cytoplasmic GFP signal in the genome-wide localization screen; consistent with a soluble enzyme acting on cytosolic eEF2. Supporting Evidence: PMID:14562095 chromosomally tagged green fluorescent protein fusion file:yeast/DPH5/DPH5-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14562095}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SL-0086 mapping of the cytoplasmic localization from Huh et al.; correct. Supporting Evidence: file:yeast/DPH5/DPH5-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14562095}. |
| GO:0005829 cytosol | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways default cytosol compartment for the Dph5 reactions. Here the default is appropriate: Dph5 is a soluble cytoplasmic protein that modifies cytosolic eEF2, and no organellar pool is reported. Supporting Evidence: file:yeast/DPH5/DPH5-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14562095}. PMID:1508200 shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant |
| GO:0008168 methyltransferase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro tetrapyrrole methylase domain mapping to generic methyltransferase activity. Reason: True but much less informative than GO:0141133 diphthine methyl ester synthase activity, which the gene already carries with experimental support. Supporting Evidence: PMID:1508200 sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases |
| GO:0017183 protein histidyl modification to diphthamide | IDA PMID:3042777 Biosynthesis of diphthamide in Saccharomyces cerevisiae. Par... | ACCEPT | Summary: Chen & Bodley (1988) showed that the dph5 mutant eEF2 is converted in vitro to the toxin-sensitive form by a SAM:EF-2 methyltransferase and that the labelled product is diphthine. Supporting Evidence: PMID:3042777 evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase PMID:3042777 Hydrolysis of labeled EF-2 yielded diphthine |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000120 | ACCEPT | Summary: InterPro IPR004551 (Dph5 family) / UniPathway UPA00559 mapping to diphthamide formation; correct for this enzyme. Supporting Evidence: file:yeast/DPH5/DPH5-uniprot.txt catalyzes four methylations of the modified target histidine residue in |
| GO:0017183 protein histidyl modification to diphthamide | IMP PMID:1508200 DPH5, a methyltransferase gene required for diphthamide bios... | ACCEPT | Summary: Mattheakis et al. (1992) cloned DPH5 by complementing the methyltransferase-step mutant; the null lacks diphthamide-dependent toxin sensitivity. Supporting Evidence: PMID:1508200 (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis PMID:1508200 shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant |
| GO:0017183 protein histidyl modification to diphthamide | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways RCA placing DPH5 in the diphthamide biosynthesis pathway (PWY-6482-1); the pathway assignment is correct even though the reaction product is outdated. Supporting Evidence: file:yeast/DPH5/DPH5-uniprot.txt catalyzes four methylations of the modified target histidine residue in PMID:24739148 Yeast Dph5 catalyzes the methylation of the amino and the carboxylate groups of ACP, generating methylated diphthine (4a). |
| GO:0141133 diphthine methyl ester synthase activity | IDA PMID:24739148 Dph7 catalyzes a previously unknown demethylation step in di... | ACCEPT | Summary: Lin et al. (2014) reconstituted Dph5 with SAM and ACP-modified eEF2 from dph5 cells and detected monomethyl-ACP, diphthine and methylated diphthine (methyl ester) by LC-MS, establishing the methyl ester as the Dph5 product. Supporting Evidence: PMID:24739148 Yeast Dph5 catalyzes the methylation of the amino and the carboxylate groups of ACP, generating methylated diphthine (4a). PMID:24739148 yeast Dph5 catalyzes tetramethylation of the ACP group |
| GO:0141133 diphthine methyl ester synthase activity | IDA PMID:3042777 Biosynthesis of diphthamide in Saccharomyces cerevisiae. Par... | ACCEPT | Summary: Partial purification of the SAM:EF-2 methyltransferase that converts dph5 eEF2; the 1988 assay reported diphthine after acid hydrolysis, which would also hydrolyse the methyl ester, so the data are consistent with the later methyl-ester assignment. Curators mapped it to the current term. Supporting Evidence: PMID:3042777 evidence is presented that the modifying enzyme is a specific S-adenosylmethionine:EF-2 methyltransferase PMID:24739148 Yeast Dph5 catalyzes the methylation of the amino and the carboxylate groups of ACP, generating methylated diphthine (4a). |
| GO:0141133 diphthine methyl ester synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RHEA:42652 / EC 2.1.1.314 mapping (UniProt catalytic activity, diphthine methyl ester synthase); correct for yeast Dph5. Supporting Evidence: file:yeast/DPH5/DPH5-uniprot.txt catalyzes four methylations of the modified target histidine residue in |
| GO:0141133 diphthine methyl ester synthase activity | IMP PMID:1508200 DPH5, a methyltransferase gene required for diphthamide bios... | ACCEPT | Summary: Recombinant Dph5 methylates eEF2 from the dph5 mutant, and the null mutant lacks the methyltransferase step. Supporting Evidence: PMID:1508200 shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant PMID:1508200 (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis |
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Download this section (compressed HTML)Q: Why does eukaryotic Dph5 esterify the carboxylate (requiring Dph7 to remove it) when archaeal Dph5 does not; is the methyl ester a regulatory checkpoint in vivo?
Experiment: Quantify methyl-ester versus diphthine versus diphthamide eEF2 by targeted LC-MS across growth phases and stresses, and test Dph5 active-site mutants that retain N-methylation but lose O-methylation for growth, frameshifting and toxin phenotypes.
Hypothesis: The O-methyl ester step is a regulated gate on diphthamide completion
Type: targeted proteomics / mutagenesis
Methyltransferase required for synthesis of diphthamide; diphthamide is a modified histidine residue of translation elongation factor 2 (Eft1p or Eft2p); not essential for viability; GFP-Dph5p fusion protein localizes to the cytoplasm
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