DPH6 (YLR143W) encodes diphthine-ammonia ligase (diphthamide synthetase, EC 6.3.1.14), the enzyme of the final step of diphthamide biosynthesis on translation elongation factor 2 (eEF2). Its N-terminal adenine nucleotide alpha hydrolase-like (ATP pyrophosphatase, SGGKDS P-loop-like motif) domain uses ATP and free ammonium (not glutamine) to amidate the carboxylate of diphthine on eEF2 His699, releasing AMP and diphosphate; the C-terminal region resembles the RutC/YjgF family. Dph6 binds eEF2 and acts after Dph7 has removed the methyl ester installed by Dph5. dph6 null cells accumulate diphthine-modified eEF2, are resistant to sordarin and diphtheria toxin, and show increased -1 ribosomal frameshifting. The human ortholog ATPBD4/DPH6 fully complements the yeast deletion. Dph6 is a cytoplasmic protein.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | HDA PMID:14562095 Global analysis of protein localization in budding yeast. | ACCEPT | Summary: Cytoplasmic GFP localization in the genome-wide screen; consistent with an enzyme modifying cytosolic eEF2. Supporting Evidence: PMID:14562095 chromosomally tagged green fluorescent protein fusion file:yeast/DPH6/DPH6-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14562095}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt SL-0086 mapping of the Huh et al. cytoplasmic localization; correct. Supporting Evidence: file:yeast/DPH6/DPH6-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:14562095}. |
| GO:0017178 diphthine-ammonia ligase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBA from the DUF71/Dph6 family node (with human ATPBD4/DPH6 and yeast Dph6 as experimental members); correct and consistent with complementation of the yeast null by human ATPBD4. Supporting Evidence: PMID:23169644 We found that yeast protein YLR143W is the diphthamide synthetase catalyzing the last amidation step using ammonium and ATP. PMID:23169644 The previously uncharacterized human gene ATPBD4 is the ortholog of yeast YLR143W and fully rescues the deletion of YLR143W in yeast. |
| GO:0017178 diphthine-ammonia ligase activity | IDA PMID:23169644 Chemogenomic approach identified yeast YLR143W as diphthamid... | ACCEPT | Summary: Purified His6-YLR143W amidated diphthine-containing eEF2 in vitro with ATP and NH4Cl (not glutamine), producing AMP. Supporting Evidence: PMID:23169644 diphthamide was formed when ammonium was used as the nitrogen source in the presence of both ATP and YLR143W PMID:23169644 AMP was found to be the major product of the diphthine amidation reaction |
| GO:0017178 diphthine-ammonia ligase activity | IEA GO_REF:0000120 | ACCEPT | Summary: RHEA:19753 / EC 6.3.1.14 mapping from the UniProt catalytic activity; correct. Supporting Evidence: file:yeast/DPH6/DPH6-uniprot.txt Reaction=diphthine-[translation elongation factor 2] + NH4(+) + ATP = |
| GO:0017178 diphthine-ammonia ligase activity | IMP PMID:23169644 Chemogenomic approach identified yeast YLR143W as diphthamid... | ACCEPT | Summary: Deletion of YLR143W abolishes diphthamide formation, supporting it as the ligase together with the in vitro data in the same paper. Supporting Evidence: PMID:23169644 We also confirmed the deficiency of diphthamide biosynthesis in Ξylr143w strain using an in vivo assay. PMID:23169644 We found that yeast protein YLR143W is the diphthamide synthetase catalyzing the last amidation step using ammonium and ATP. |
| GO:0017183 protein histidyl modification to diphthamide | IBA GO_REF:0000033 | ACCEPT | Summary: PANTHER IBA for diphthamide formation from the Dph6 family node; correct. Supporting Evidence: PMID:23169644 The previously uncharacterized human gene ATPBD4 is the ortholog of yeast YLR143W and fully rescues the deletion of YLR143W in yeast. |
| GO:0017183 protein histidyl modification to diphthamide | IDA PMID:23169644 Chemogenomic approach identified yeast YLR143W as diphthamid... | ACCEPT | Summary: In vitro reconstitution of the final amidation step with purified Dph6. Supporting Evidence: PMID:23169644 diphthamide was formed when ammonium was used as the nitrogen source in the presence of both ATP and YLR143W |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway UPA00559 mapping; correct. Supporting Evidence: file:yeast/DPH6/DPH6-uniprot.txt FUNCTION: Amidase that catalyzes the last step of diphthamide |
| GO:0017183 protein histidyl modification to diphthamide | IMP PMID:23169644 Chemogenomic approach identified yeast YLR143W as diphthamid... | ACCEPT | Summary: Diphthamide deficiency of the ylr143w deletion strain in vivo. Supporting Evidence: PMID:23169644 We also confirmed the deficiency of diphthamide biosynthesis in Ξylr143w strain using an in vivo assay. |
| GO:0017183 protein histidyl modification to diphthamide | IMP PMID:23468660 The amidation step of diphthamide biosynthesis in yeast requ... | ACCEPT | Summary: Uthman et al. (2013): dph6 mutants accumulate diphthine-modified eEF2 and share the toxin/sordarin resistance of dph1-dph5. Supporting Evidence: PMID:23468660 dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2 PMID:23468660 Dph6 contains an essential adenine nucleotide hydrolase domain and binds to eEF2 |
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Download this section (compressed HTML)Q: What is the function of the C-terminal RutC/YjgF-like region of Dph6; is it needed for eEF2 binding or for ammonia handling?
Experiment: Express Dph6 N-terminal ATP-pyrophosphatase domain alone versus full length; compare eEF2 co-immunoprecipitation, in vitro amidation kinetics, and complementation of dph6 sordarin resistance.
Hypothesis: The RutC-like C-terminal domain contributes to eEF2 recognition
Type: domain truncation / biochemistry
Diphthamide synthetase; catalyzes the last amidation step of diphthamide biosynthesis using ammonium and ATP; evolutionarily conserved in eukaryotes; dph6 mutants exhibit diphthine accumulation and resistance to sordarin, which is indicative of defects in diphthamide formation on EF2; green fluorescent protein (GFP)-tagged protein localizes to the cytoplasm; DPH6/YLR143W is not an essential gene
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