NADK2 is the mitochondrial NAD(+) kinase (EC 2.7.1.23) of the NAD kinase family (PANTHER PTHR13158:SF5, NAD kinase 2, mitochondrial). It transfers the gamma-phosphate of ATP (or, in vitro, of inorganic polyphosphate) to NAD(+) to form NADP(+), and is the source of the mitochondrial NADP(H) pool, separate from the cytosolic pool made by NADK. The NADPH derived from this pool is used by NADP-dependent matrix oxidoreductases, such as 2,4-dienoyl-CoA reductase of polyunsaturated fatty acid oxidation and the first step of lysine degradation, and by mitochondrial antioxidant systems. The protein is imported into mitochondria by an N-terminal targeting sequence and acts as a soluble enzyme that is thought to form a homodimer. The donkey entry A0A8C4M4D0 (538 aa) is the full-length product of the donkey NADK2 gene. It aligns without gaps to the whole canonical human NADK2 sequence (96.8% identity), keeps the GGDG motif and the segment that carries the predicted NAD(+)-binding positions, and begins 96 residues upstream of the methionine that aligns to human Met1. All functional statements for the donkey protein rest on homology to the experimentally characterised human ortholog in the same subfamily; none has been tested in donkey.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003951 NAD+ kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: NAD+ kinase activity from the combined IEA pipeline (ARBA, EC mapping and the PANTHER node). The donkey gene is in PANTHER subfamily PTHR13158:SF5 with human NADK2 (Q4G0N4), whose purified recombinant protein forms NADP+ from NAD+ using ATP or polyphosphate (PMID:23212377). PAINT curators placed NAD+ kinase activity at PTN004406956, seeded by human and mouse NADK2. Reason: This is the defining activity of the NADK2 subfamily, and the donkey protein sits in the same subfamily as the characterised human enzyme. A0A8C4M4D0 is the full-length model and keeps every sequence feature that the activity is predicted to need. It aligns gap-free to human canonical NADK2, keeps the GGDG motif (donkey 255-258), and keeps the positions equivalent to human Ser306/Lys317/Trp319 (donkey S402/K413/W415), which PMID:23212377 maps to the NAD+-binding residues of bacterial NAD kinase. The activity is inferred for donkey, not measured. Supporting Evidence: PMID:23212377 showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors file:human/NADK2/NADK2-uniprot.txt Mitochondrial NAD(+) kinase that phosphorylates NAD(+) to file:EQUAS/NADK2__A0A8C4M4D0/NADK2__A0A8C4M4D0-uniprot.txt PANTHER; PTHR13158:SF5; NAD KINASE 2, MITOCHONDRIAL; 1. file:EQUAS/NADK2/NADK2-bioinformatics/RESULTS-rereview.md A0A8C4M4D0 (538 aa) aligns gap-free to the whole human canonical sequence |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Mitochondrion. Human NADK2 localises to mitochondria in HEK293A cells by immunofluorescence and by cell fractionation, and its N-terminal 62 residues are a functional mitochondrial targeting sequence (PMID:23212377). PAINT placed mitochondrion at PTN000994636, seeded by human and mouse NADK2. Reason: Mitochondrion is the level of localisation that was established experimentally for the human ortholog in the same subfamily, and PAINT propagated it. The donkey sequence keeps the region equivalent to the human transit peptide (donkey 97-158). In every equid gene model this region lies 96 residues from the N-terminus, so the donkey start site is uncertain, but this does not cast doubt on the gene-level localisation. The more specific matrix location (next row) is inferred rather than assayed, so this row is kept as it stands. Supporting Evidence: PMID:23212377 confirming that C5orf33 protein is a mitochondrial NADK, and that its N-terminal 62 residues contain a functional mitochondrial-targeting sequence file:human/NADK2/NADK2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:23212377}. file:EQUAS/NADK2/NADK2-bioinformatics/RESULTS-rereview.md the region corresponding to the human transit peptide (human 1-62) is internal (donkey 97-158) in these models |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000117 | ACCEPT | Summary: Mitochondrial matrix (ARBA). NADK2 is a soluble enzyme with no transmembrane segment. It is imported by a cleavable N-terminal targeting sequence, and it supplies NADP(H) to matrix NADP-dependent enzymes such as dienoyl-CoA reductase and the first enzyme of lysine degradation (PMID:24847004). Reason: The matrix is the compartment in which NADK2 is expected to act. The NADP-dependent enzymes that become deficient when NADK2 is lost are matrix oxidoreductases, and the protein has a transit peptide but no membrane anchor. This placement has not been confirmed by submitochondrial fractionation in any species, so it is the most specific location that is reasonably supported rather than a measured one. Supporting Evidence: PMID:24847004 DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency PMID:23212377 confirming that C5orf33 protein is a mitochondrial NADK, and that its N-terminal 62 residues contain a functional mitochondrial-targeting sequence |
| GO:0019674 NAD+ metabolic process | IEA GO_REF:0000120 | MODIFY | Summary: NAD+ metabolic process from the combined IEA pipeline (InterPro IPR017437 and the PANTHER node). It matches the PAINT IBD for this term at PTN004406956, seeded by human NADK2. The enzyme consumes NAD+ to make NADP+. Reason: The term is true but general. The step that NADK2 itself catalyses is the phosphorylation of NAD+ to NADP+, and NAD kinase is the only enzyme that makes NADP+, so NADP+ biosynthetic process (GO:0006741) is the precise term. GO:0006741 is an is_a descendant of GO:0019674 (checked with OLS hierarchicalAncestors), and the donkey UniProt record also carries it through InterPro. The participation test is met because NADK2 performs the step. Proposed replacements: NADP+ biosynthetic process Supporting Evidence: PMID:23212377 NAD kinase is the sole NADP(+) biosynthetic enzyme PMID:24847004 NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts file:EQUAS/NADK2__A0A8C4M4D0/NADK2__A0A8C4M4D0-uniprot.txt InterPro; IPR002504; NADK. |
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Download this section (compressed HTML)Q: Does donkey NADK2 start translation at Met1 of the Ensembl model or at Met97, which aligns to human Met1? The 96-residue upstream extension is shared by all equid models; if it is translated, is the internal transit-peptide-equivalent region (donkey 97-158) still recognised for import?
Q: Human NADK2 is a crystallographically confirmed homodimer held together by an insert reported as conserved in animal mitochondrial NAD kinases (PMID:35733246, PMID:35868311), but PAINT has not placed homodimerization in PTHR13158 and no row for it reaches this donkey entry. Should PAINT place it at the animal node once conservation of the insert has been mapped across the family (see the PTHR13158 family review)?
Experiment: Express donkey NADK2 (residues from Met97, with and without the upstream extension) in the Saccharomyces cerevisiae utr1 yef1 pos5 NADK triple mutant and as a purified recombinant protein, to confirm ATP-dependent NAD+ kinase activity and measure its kinetics against human NADK2.
Type: complementation and enzyme assay
Experiment: Map the native N-terminus of donkey NADK2 by mass spectrometry of mitochondrial fractions from donkey tissue (for example liver), and determine its submitochondrial location by protease protection or fractionation.
Type: proteomics and subcellular fractionation
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