Gpdh3 (CG43343) encodes a large (about 1,300 residues), uncharacterized Drosophila paralog of the cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase Gpdh1. Its first ~340 residues form a glycerol-3-phosphate dehydrogenase fold (NAD-binding Rossmann domain plus the helical C-terminal domain), followed by a long, largely disordered C-terminal extension with no recognized domain. The gene is one of two long-branch, testis-biased Gpdh paralogs that arose in the higher Diptera. Gpdh3 diverges at residues essential for the enzyme in its relatives: its NAD-binding glycine loop reads GxExxG instead of GxGxxG in all Drosophila orthologs, and in the melanogaster group the lysine that serves as the proton acceptor in human GPD1 is replaced by threonine. Whether D. melanogaster Gpdh3 still interconverts dihydroxyacetone phosphate and glycerol-3-phosphate, binds NAD, or has taken on a different role in the male germline has not been tested. The gene makes three products (PA 1304 aa, PB 1291 aa, PC 1303 aa) that differ only by a 14-residue segment just after the C-terminal GPDH domain; E1JIT1 is the PB product.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | UNDECIDED | Summary: Process inferred from the GPDH C-terminal domain signature, but whether the D. melanogaster protein still catalyses the GPDH reaction is unresolved. Reason: InterPro2GO assigns this generic process from family membership. Gpdh3 does sit in PANTHER PTHR11728:SF8 with Gpdh1, but the D. melanogaster protein has lost the GPD1 proton-acceptor lysine (T205) and carries a GxExxG NAD loop. Participation in any metabolic process therefore rests on untested catalytic activity. This row is handled the same way as the more specific glycerol-3-phosphate metabolic process row. It is not replaced, because the specific term is equally unverified. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md Catalytic-loop window file:DROME/Gpdh3/Gpdh3-uniprot.txt DR InterPro; IPR006109; G3P_DH_NAD-dep_C. |
| GO:0006072 glycerol-3-phosphate metabolic process | IEA GO_REF:0000002 | UNDECIDED | Summary: Ancestral process of the GPDH family; not established for D. melanogaster Gpdh3, whose catalytic lysine is replaced. Reason: The gene (PANTHER leaf Q8IN06, Gpdh3-PA) inherits a positive IBA for this term from PTN000198006, and PAINT placed a NOT only on the CG34149 leaf, not on Gpdh3. That is a deliberate phylogenetic judgment and is the strongest argument for keeping the term. Against it, there is target-specific evidence of divergence that a node-level inference cannot capture. The GPD1 K204 proton acceptor is T205 in D. melanogaster and in the other melanogaster-group orthologs, although obscura-group and willistoni orthologs retain the lysine. The NAD-binding loop GSGNWG is GAEGWG throughout the Gpdh3 lineage. No assay of any Gpdh3 product exists. The term is not removed, but it cannot be accepted until activity is measured. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md The catalytic lysine is retained (`CGTLKD`) in D. pseudoobscura, D. persimilis, D. madeirensis, file:DROME/Gpdh3/Gpdh3-uniprot.txt DR InterPro; IPR017751; G3P_DH_NAD-dep_euk. |
| GO:0006650 glycerophospholipid metabolic process | IEA GO_REF:0000041 | MARK AS OVER ANNOTATED | Summary: Pathway-level extrapolation from the EC assignment; no evidence links Gpdh3 to phospholipid metabolism. Reason: The UniPathway mapping (alpha-glycerophosphate cycle) follows from an ARBA/UniRule EC assignment, and the protein's catalytic activity itself is doubtful. Neither the PANTHER IBA set nor any experiment places Gpdh3 in glycerophospholipid metabolism. Even for active cytosolic GPDHs, supplying glycerol-3-phosphate to lipid synthesis is a metabolic context, not a role specific to this paralog. The testis-biased expression gives no further support for the term. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md Canonical NAD-dependent PMID:33083121 Glycerol 3 phosphate dehydrogenaseGpdh1cytosolicmuscle specific0Gpdh211Gpdh311 |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | UNDECIDED | Summary: Generic NAD(P)-dependent oxidoreductase activity is inferred from the N-terminal domain. Gpdh3 has lost both the catalytic base and the NAD-loop glycine, so the activity is unverified. Reason: This is the broad parent of GO:0141152 and has the same evidential problem. The Rossmann fold is present, but the loss of the proton-acceptor lysine and the GxExxG NAD loop make the canonical hydride-transfer chemistry doubtful in D. melanogaster. The row is not modified to the specific term, because the specific activity is equally unestablished. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md 204 (ACT_SITE, proton acceptor) | K204 | K206 | T205 |
| GO:0042803 protein homodimerization activity | IEA GO_REF:0000002 | UNDECIDED | Summary: Homodimerization of the long Gpdh3 product has not been examined. Reason: The IPR017751 mapping transfers the dimeric assembly of short eukaryotic GPDHs. Gpdh3 keeps the C-terminal helical domain, which in GPDHs forms the dimer interface. However, it carries a roughly 950-residue disordered extension and has diverged substantially (about 45% identity to Gpdh1 over the core). No assembly data exist for any Gpdh3 product. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-uniprot.txt FT DOMAIN 194..312 file:DROME/Gpdh3/Gpdh3-uniprot.txt FT REGION 924..1106 |
| GO:0046168 glycerol-3-phosphate catabolic process | IEA GO_REF:0000120 | MARK AS OVER ANNOTATED | Summary: The direction-specific catabolic process is an extrapolation from a reversible reaction written in the oxidative direction, for a protein whose activity is itself unverified. Reason: The UniRule maps the EC 1.1.1.8 reaction to glycerol-3-phosphate catabolism. Cytosolic NAD-GPDHs generally act in the DHAP-to-glycerol-3-phosphate direction, and PAINT propagated only the direction-neutral glycerol-3-phosphate metabolic process (PTN000198006) to Gpdh3, not a catabolic term. On top of that, the D. melanogaster protein lacks the catalytic lysine. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md Canonical NAD-dependent |
| GO:0051287 NAD binding | IEA GO_REF:0000120 | UNDECIDED | Summary: NAD binding is predicted from the Rossmann domain, but the GxGxxG glycine loop that contacts the cofactor pyrophosphate is GxExxG in every Gpdh3 ortholog. Reason: Several NAD contact positions are kept (K120, N270, K296), and Rossmann-fold pseudoenzymes can still bind nucleotide, so binding is plausible. The glycine-to-glutamate change in the first glycine-rich loop is conserved across the Gpdh3 lineage and is likely to alter cofactor binding. No binding data exist, so the annotation is neither accepted nor removed. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md NAD loop: human `GSGNWG`, Gpdh1 `GSGNWG`, Gpdh3 `GAEGWG`. file:DROME/Gpdh3/Gpdh3-uniprot.txt FT DOMAIN 6..173 |
| GO:0141152 glycerol-3-phosphate dehydrogenase (NAD+) activity | IEA GO_REF:0000120 | UNDECIDED | Summary: Ancestral GPDH activity of the family; the D. melanogaster protein has lost the proton-acceptor lysine, so activity is untested and doubtful. Reason: The previous review accepted this term from domain completeness. Re-examination shows that domain presence is not residue conservation. Gpdh3 is a PTHR11728:SF8 member, and the gene receives a positive PAINT IBA for the parent NAD(P)+ activity (GO:0047952) via PTN000919759. That is a real phylogenetic argument that the lineage was ancestrally an active GPDH, and obscura-group and willistoni orthologs still retain the catalytic lysine. In D. melanogaster and the rest of the melanogaster group, the residue aligned with the human GPD1 proton acceptor K204 is threonine, and the NAD-loop glycine is glutamate. The substitution is present in cDNA-derived entries as well as the genome model, so it is not a sequence error. This lineage-specific loss is the kind of target-specific divergence that an IBA node cannot see. The annotation is not removed, because no assay has shown inactivity. It should be resolved experimentally. Supporting Evidence: file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md 204 (ACT_SITE, proton acceptor) | K204 | K206 | T205 file:DROME/Gpdh3/Gpdh3-bioinformatics/RESULTS-rereview.md In D. melanogaster the threonine is present in all cDNA- and genome-derived entries file:DROME/Gpdh3/Gpdh3-uniprot.txt DR PANTHER; PTHR11728:SF8; GLYCEROL-3-PHOSPHATE DEHYDROGENASE [NAD(+)]-RELATED; 1. file:DROME/Gpdh3/Gpdh3-deep-research-falcon.md No primary publication directly characterizing CG43343/E1JIT1 was identified |
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Download this section (compressed HTML)Q: Does D. melanogaster Gpdh3 retain any NAD-dependent glycerol-3-phosphate dehydrogenase activity despite the K-to-T substitution at the GPD1 proton-acceptor position, and does the obscura-group ortholog that retains the lysine?
Q: Should the PAINT IBA for GO:0047952 propagated to Gpdh3 from PTN000919759 be restricted given the melanogaster-group loss of the catalytic lysine (compare the NOT already placed on CG34149)?
Q: What is the function of the ~950-residue disordered C-terminal extension in the male germline?
Experiment: Express the GPDH cores (residues ~1-340) of D. melanogaster Gpdh3, a T205K revertant and the D. pseudoobscura ortholog, with fly Gpdh1 as a positive control; assay NADH-dependent DHAP reduction and NAD+-dependent G3P oxidation, and measure NAD(H) binding by fluorescence or ITC.
Hypothesis: The GPDH core of D. melanogaster Gpdh3 has lost DHAP reductase / G3P dehydrogenase activity because of the T205 substitution, while the D. pseudoobscura ortholog (lysine retained) is active.
Experiment: Determine Gpdh3 protein localization in testis with a tagged knock-in, and test male fertility and sperm metabolism in a CRISPR null.
Hypothesis: Gpdh3 acts in spermatogenesis.
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