Zebrafish dph2 encodes the DPH2 subunit of the 2-(3-amino-3-carboxypropyl)histidine synthase complex, which carries out the first and committed step of diphthamide biosynthesis: transfer of a 3-amino-3-carboxypropyl group from S-adenosyl-L-methionine onto a specific histidine of translation elongation factor 2 (eEF2). In eukaryotes the enzyme is a Dph1-Dph2 heterodimer acting with Dph3 and an NADH-dependent reductase (PMID:24422557, PMID:29590073). Each subunit binds a [4Fe-4S] cluster coordinated by three cysteines drawn from three different domains (PMID:20559380); the cluster in Dph2 reduces the catalytic cluster in Dph1, which cleaves SAM to generate the ACP radical, so Dph2 contributes to an activity that belongs to the heterodimer rather than possessing it alone. Diphthamide is the residue ADP-ribosylated by diphtheria toxin and supports translational fidelity; in humans, biallelic DPH1 or DPH2 variants that abolish the modification cause a developmental disorder with growth retardation and craniofacial anomalies (PMID:32576952). Zebrafish dph2 itself has not been functionally characterized; all assignments here come from orthology.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0017183 protein histidyl modification to diphthamide | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the DPH1/DPH2 family node (PTN000079945), seeded by both yeast subunits (DPH1 S000001674, DPH2 S000001365), two mouse genes and human DPH2. It asserts that participation in diphthamide formation on eEF2 arose before the fungal/animal split and is inherited by zebrafish dph2. Reason: The node placement is secure: the DPH1/DPH2 family exists only to make diphthamide, the pathway and the eEF2 substrate are conserved from archaea to vertebrates (PMID:29590073), and zebrafish dph2 is a full-length member of the DPH2 subfamily (UniProt SIMILARITY). dph2 does part of the work of the process - it supplies the electron-donating cluster for the ACP transfer - so participation, not mere requirement, is satisfied. Core process. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Required for the first step of diphthamide biosynthesis, a PMID:29590073 The enzyme that performs this reaction is a Dph2 (diphthamide biosynthesis protein 2) homodimer in archaea, such as Pyrococcus horikoshii (PhDph2) (5) or a Dph1-Dph2 heterodimer in eukaryotes (6). PMID:32576952 The gene products DPH1 and DPH2 are components of a heterodimeric enzyme complex that mediates the first step of the posttranslational dipht |
| GO:0017183 protein histidyl modification to diphthamide | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation (InterPro IPR010014/IPR016435, ARBA, UniPathway UPA00559) assigning the diphthamide pathway from domain content. Reason: The InterPro families are diphthamide-synthesis specific rather than generic, so the mapping is informative here, and it agrees with the IBA and ISS rows and with the UniProt PATHWAY line. Same action as those rows. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Required for the first step of diphthamide biosynthesis, a |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO mapping of IPR016435 (DPH1/DPH2 family) to the ACP synthase activity with an enables qualifier, i.e. asserting that dph2 by itself has the activity. The family signature cannot distinguish the catalytic Dph1 subunit from the electron-donating Dph2 subunit. Reason: The activity is real but is not one dph2 has on its own. Dph2 is one of two subunits: its own [4Fe-4S] cluster feeds electrons to the catalytic cluster in Dph1, which performs the SAM cleavage (UniProt COFACTOR/FUNCTION; PMID:24422557, PMID:29590073). The activity therefore belongs to the Dph1-Dph2 heterodimer, and only the contributes_to form of the annotation states that correctly. The parallel ISS row states exactly this with contributes_to and is accepted; this row is kept but flagged, because the enables form over-states a single subunit as the whole enzyme. The two rows differ only in qualifier strength, which is why their actions differ. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Facilitates the reduction of the catalytic iron-sulfur cluster found in the dph1 subunit (By similarity). PMID:24422557 suggesting that Dph1 and Dph2 form a heterodimer. PMID:29590073 Dph2 binds an essential [4Fe-4S] cluster and is thought to use the reduced (1+) state of the cluster to cleave the CΞ³,Met-S bond of SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. |
| GO:0120513 2-(3-amino-3-carboxypropyl)histidine synthase complex | ISS GO_REF:0000024 | ACCEPT | Summary: Manual ISS transfer from yeast DPH2 (UniProtKB:P32461) of membership in the ACP synthase complex. UniProt records the complex as dph1, dph2, dph3 and a NADH-dependent reductase; the Dph1-Dph2 core was shown to be a heterodimer carrying [4Fe-4S] clusters (PMID:24422557). Reason: Complex membership is the level at which dph2 acts, and it is what makes the contributes_to form of GO:0090560 appropriate. The yeast donor has this complex assignment by direct experiment, and the zebrafish protein is a conserved DPH2-subfamily member (UniProt SIMILARITY). Core. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Component of the 2-(3-amino-3-carboxypropyl)histidine synthase complex composed of dph1, dph2, dph3 and a NADH-dependent reductase. PMID:24422557 suggesting that Dph1 and Dph2 form a heterodimer. |
| GO:0017183 protein histidyl modification to diphthamide | ISS GO_REF:0000024 | ACCEPT | Summary: Manual ISS transfer from budding yeast DPH2 (UniProtKB:P32461), whose loss abolishes diphthamide on eEF2. Third row for this term (with IBA and IEA), all asserting the same participation. Reason: The yeast donor is the experimentally defined subunit, and the human orthologs carry disease variants that abolish diphthamide synthesis on eEF2 (PMID:32576952), so the function is retained across the clade that includes zebrafish. Accepted, consistently with the IBA and IEA rows for this term. Supporting Evidence: PMID:32576952 We analyzed the catalytic activity of the human DPH2 variants and their yeast homologues, revealing both variants drastically reduce or abolish diphthamide biosynthesis on eEF2. file:DANRE/dph2/dph2-uniprot.txt Required for the first step of diphthamide biosynthesis, a |
| GO:0051539 4 iron, 4 sulfur cluster binding | ISS GO_REF:0000024 | ACCEPT | Summary: Manual ISS transfer from yeast DPH2 (UniProtKB:P32461) of [4Fe-4S] cluster binding. The cluster is coordinated by three cysteines contributed from three different domains, the arrangement first seen in the archaeal PhDph2 structure (PMID:20559380), and UniProt annotates one cluster per subunit for the zebrafish protein. Reason: This is a genuine molecular function of dph2 itself, not of the complex: its cluster reduces the catalytic cluster in Dph1 (UniProt COFACTOR), and Dph3 delivers the electron to it (PMID:24422557, PMID:34154323). Accepted as a core molecular function; the cysteine ligands are conserved in the zebrafish sequence but have not been tested there. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Binds 1 [4Fe-4S] cluster per subunit. The cluster facilitates the PMID:20559380 The X-ray crystal structure of PhDph2 revealed an intriguing constellation of three conserved cysteine residues -- each from a different structural domain -- suggestive of an iron-sulfur cluster. PMID:20559380 A crystal structure of reconstituted PhDph2 along with UV-Vis, EPR, and MΓΆssbauer spectroscopies confirmed the presence of a [4Fe-4S] cluster. PMID:24422557 donor to reduce the Fe-S cluster in Dph1-Dph2. |
| GO:0090560 2-(3-amino-3-carboxypropyl)histidine synthase activity | ISS GO_REF:0000024 | ACCEPT | Summary: Manual ISS transfer from yeast DPH2 (UniProtKB:P32461) of the ACP synthase activity with the contributes_to qualifier, i.e. dph2 is a subunit of the enzyme that has this activity rather than an independent catalyst. Reason: Graded on the weaker claim the qualifier makes, which is exactly right for this protein. Dph2 is one of two subunits: its own [4Fe-4S] cluster feeds electrons to the catalytic cluster in Dph1, which performs the SAM cleavage (UniProt COFACTOR/FUNCTION; PMID:24422557, PMID:29590073). The activity therefore belongs to the Dph1-Dph2 heterodimer, and only the contributes_to form of the annotation states that correctly. This row is kept ACCEPT while the GO:0090560 IEA row, which asserts enables, is marked over-annotated; the difference between the two rows is the qualifier, not the biology. Supporting Evidence: PMID:29590073 The enzyme that performs this reaction is a Dph2 (diphthamide biosynthesis protein 2) homodimer in archaea, such as Pyrococcus horikoshii (PhDph2) (5) or a Dph1-Dph2 heterodimer in eukaryotes (6). PMID:24422557 suggesting that Dph1 and Dph2 form a heterodimer. file:DANRE/dph2/dph2-uniprot.txt Facilitates the reduction of the catalytic iron-sulfur cluster found in the dph1 subunit (By similarity). |
| GO:0005829 cytosol | ISS | NEW | Summary: Proposed cellular component. GOA lists no cellular-component annotation for zebrafish dph2, but the Dph1-Dph2 complex modifies eEF2, a cytosolic translation elongation factor, and the orthologs carry the location: yeast DPH2 (P32461) has GO:0005737 by HDA from a genome-wide GFP localization study, and human DPH2 (Q9BQC3) has GO:0005829 cytosol. Reason: Proposed on the same ISS basis as the other rows. The human ortholog DPH2 (Q9BQC3) is annotated to cytosol (GO:0005829), the yeast donor to cytoplasm, the substrate eEF2 is a cytosolic translation factor, and dph2 has no signal peptide, transmembrane segment or organellar targeting sequence in UniProt. Cytosol is therefore the informative term the evidence supports; the parent cytoplasm would be true but uninformative, the same granularity standard applied elsewhere in this batch. It is a location, not a process claim, so it asserts only where the accepted complex and cluster-binding functions are exercised. No zebrafish localization experiment exists, which is listed under suggested_experiments. Supporting Evidence: file:DANRE/dph2/dph2-uniprot.txt Component of the 2-(3-amino-3-carboxypropyl)histidine synthase complex composed of dph1, dph2, dph3 and a NADH-dependent reductase. file:DANRE/dph2/dph2-uniprot.txt Dph1 and dph2 transfer a 3-amino-3- 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** |
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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
Hypothesis: Zebrafish dph2 is required for diphthamide formation on eEF2 in vivo.
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
Hypothesis: Zebrafish Dph2 binds a [4Fe-4S] cluster through the three conserved cysteines and supports Dph1-Dph2 ACP transferase activity.
Experiment: Determine the subcellular distribution of tagged or endogenous dph2 in zebrafish embryos by fractionation or imaging, to replace the ortholog-based cytoplasm proposal with direct evidence.
Hypothesis: Zebrafish dph2 is a cytosolic protein, as its yeast and human orthologs are.
Type: localization
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