Dph2 is a subunit of the Dph1-Dph2 heterodimeric complex that catalyzes the first step of diphthamide biosynthesis, the transfer of a 3-amino-3-carboxypropyl (ACP) group from S-adenosyl-L-methionine to a conserved histidine of eukaryotic translation elongation factor 2 (eEF2). Dph2 binds a [4Fe-4S] cluster that facilitates reduction of the catalytic iron-sulfur cluster in the Dph1 subunit. Diphthamide is a unique post-translational modification of eEF2 that ensures translational fidelity and is the target of diphtheria toxin ADP-ribosylation. Loss of DPH2 function in humans causes diphthamide-deficiency syndrome, an autosomal recessive developmental disorder.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0017183 protein histidyl modification to diphthamide | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference from orthologs in yeast (DPH1, DPH2) and mouse correctly places dph2 in the diphthamide biosynthesis pathway. The DPH1-DPH2 heterodimer catalyzes the first step of diphthamide synthesis on eEF2, a pathway conserved from archaea to vertebrates. Supporting Evidence: PMID:32576952 The gene products DPH1 and DPH2 are components of a heterodimeric enzyme complex that mediates the first step of the posttranslational diphthamide modification on the nonredundant eukaryotic translation elongation factor 2 (eEF2) file:DANRE/dph2/dph2-deep-research-falcon.md DPH2 (together with DPH1) functions in the ...first committed step... of diphthamide biosynthesis: transfer of a ...3-amino-3-carboxypropyl (ACP)... group derived from ...S-adenosylmethionine (SAM/AdoMet)... onto the ...C2 of the imidazole ring... of a conserved eEF2 histidine. This ACP addition generates an early diphthamide-pathway intermediate on eEF2 |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation from InterPro domain matches (IPR010014, IPR016435) and UniPathway correctly assigns diphthamide biosynthesis. Consistent with the well-characterized biochemical role of DPH2 family proteins. Supporting Evidence: PMID:20559380 Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping from IPR016435 (Diphthamide synthesis DPH1/DPH2 family) assigns the ACP synthase activity with an implicit enables qualifier. However, Dph2 does not independently catalyze the reaction; the radical chemistry resides in the Dph1 subunit. The ISS annotation (below) correctly uses contributes_to. This IEA annotation over-annotates by implying Dph2 independently enables the activity. Reason: The IEA annotation assigns the full enables qualifier, but Dph2 only contributes to the complex activity. The ISS annotation with contributes_to qualifier is the correct representation. Supporting Evidence: PMID:20559380 Diphthamide biosynthesis requires an organic radical generated by an iron-sulphur enzyme PMID:24422557 Yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant |
| GO:0120513 2-(3-amino-3-carboxypropyl)histidine synthase complex | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence similarity transfer from yeast DPH2 (P32461). Dph2 is a subunit of the Dph1-Dph2 heterodimer in eukaryotes, which together with Dph3 and NADH-dependent reductase forms the ACP synthase complex. Well supported by biochemical evidence in yeast and archaea. Supporting Evidence: PMID:24422557 Yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro file:DANRE/dph2/dph2-deep-research-falcon.md Modern pathway summaries indicate the initial step is catalyzed by an Fe–S DPH1/DPH2 heterodimer, with involvement of DPH3/DPH4 in supporting the reaction |
| GO:0017183 protein histidyl modification to diphthamide | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence similarity transfer from yeast DPH2 (P32461). This duplicates the IBA and IEA annotations for the same term but with ISS evidence. The annotation is correct as DPH2 is required for diphthamide biosynthesis. Supporting Evidence: PMID:32576952 The gene products DPH1 and DPH2 are components of a heterodimeric enzyme complex that mediates the first step of the posttranslational diphthamide modification on the nonredundant eukaryotic translation elongation factor 2 (eEF2) |
| GO:0051539 4 iron, 4 sulfur cluster binding | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence similarity transfer from yeast DPH2 (P32461). The zebrafish Dph2 has three conserved cysteine residues (Cys89, Cys110, Cys341) that coordinate a [4Fe-4S] cluster, as annotated in UniProt by similarity. The Dph2 cluster facilitates reduction of the catalytic cluster in Dph1 rather than directly participating in radical chemistry. Supporting Evidence: PMID:34154323 all radical S-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)3 cluster for activity file:DANRE/dph2/dph2-deep-research-falcon.md One Fe-S cluster per subunit; atypical cysteine motifs instead of canonical CX3CX2C radical-SAM motif |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Sequence similarity transfer from yeast DPH2 (P32461) with the correct contributes_to qualifier. Dph2 is required for ACP synthase activity as part of the Dph1-Dph2 complex but does not independently catalyze the radical SAM reaction. The contributes_to qualifier appropriately reflects that Dph1 houses the catalytic [4Fe-4S] cluster. Supporting Evidence: PMID:29590073 Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C-S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical PMID:24422557 Yeast Dph1 and Dph2 form a complex (Dph1-Dph2) that is equivalent to the homodimer of PhDph2 and is sufficient to catalyze the first step in vitro in the presence of dithionite as the reductant file:DANRE/dph2/dph2-deep-research-falcon.md Dph1 and Dph2 are homologous and are thought to form a ...heterodimer... that is functionally analogous to archaeal homodimeric Dph2 |
| GO:0005737 cytoplasm | ISS | NEW | Summary: Dph2 functions in the cytoplasm where it forms a complex with Dph1 to modify cytoplasmic eEF2. The diphthamide biosynthesis machinery operates on eEF2 in the cytosol, consistent with its role in translation elongation. No transmembrane domains or signal peptides are present. Reason: Cytoplasmic localization is implied by the function of the Dph1-Dph2 complex on cytosolic eEF2 but was not explicitly annotated in GOA. The BioReason SFT analysis also identified cytoplasmic localization. Supporting Evidence: file:DANRE/dph2/dph2-deep-research-bioreason-sft.md The cellular component is dictated by where eEF2 resides and where ferredoxin-coupled redox partners operate. The adaptor must access cytosolic eEF2 and the cytosolic ferredoxin/reductase chain, supporting localization to the GO:0005737 cytoplasm file:DANRE/dph2/dph2-deep-research-falcon.md diphthamide is a modification on the ...cytosolic translation factor eEF2..., so the pathway is expected to act in the ...cytosol... ...Arabidopsis DPH2 localizes to the cytosol and physically interacts with DPH1..., consistent with a cytosolic biosynthetic complex acting on cytosolic eEF2 |
Loading supporting content…
Download this section (compressed HTML)Q: Is there direct experimental evidence for dph2 function in zebrafish, or are all functional assignments based on orthology to yeast and human DPH2?
Q: Does zebrafish dph2 deficiency produce neural crest or craniofacial phenotypes analogous to those seen in mouse Dph1 knockin models?
Q: Is the [4Fe-4S] cluster in zebrafish dph2 essential for its function, and does Dph3-mediated iron donation operate similarly in fish?
Experiment: CRISPR-Cas9 knockout of dph2 in zebrafish embryos to assess developmental phenotypes, particularly craniofacial and neural crest derivatives, with comparison to dph1 mutants
Experiment: In vitro reconstitution of zebrafish Dph1-Dph2 complex to confirm ACP transferase activity and [4Fe-4S] cluster requirements using site-directed mutagenesis of the three conserved cysteine residues
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)