NADK2

UniProt ID: A0A9L0K2P4
Organism: Equus asinus
Review Status: COMPLETE
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Gene Description

NADK2 is the mitochondrial NAD(+) kinase (EC 2.7.1.23) of the NAD kinase family (PANTHER PTHR13158:SF5, NAD kinase 2, mitochondrial). It phosphorylates NAD(+) using ATP (or, in vitro, inorganic polyphosphate) to produce NADP(+), and is the source of the mitochondrial matrix NADP(H) pool. This pool supplies NADPH-dependent matrix enzymes, including 2,4-dienoyl-CoA reductase of polyunsaturated fatty acid oxidation and the first step of lysine degradation, and supports antioxidant defence; loss of NADK2 causes mitochondrial NADP(H) deficiency. The protein is imported into mitochondria by an N-terminal targeting sequence and acts as a soluble matrix enzyme. The donkey entry A0A9L0K2P4 is one of three gene models of the single donkey NADK2 gene. It corresponds to human isoform 2, lacking the 32-residue segment equivalent to human 288-319, which contains the positions mapped to the NAD(+)-binding site of bacterial NAD kinases, while retaining the GGDG motif. Like the other equid models, it also begins 96 residues upstream of the methionine that aligns to human Met1.

Existing Annotations Review

GO Term Evidence Action Reason
GO:0003951 NAD+ kinase activity
IEA
GO_REF:0000120 		ACCEPT
Summary: NAD+ kinase activity transferred by the combined IEA pipeline (ARBA, EC mapping and the PANTHER node). The donkey gene belongs to PANTHER subfamily PTHR13158:SF5 together with human NADK2 (Q4G0N4), whose purified recombinant protein forms NADP+ from NAD+ with ATP or polyphosphate (PMID:23212377), and PAINT curators placed NAD+ kinase activity at PTN004406956, seeded by human and mouse NADK2.
Reason: The activity is the defining function of the NADK2 subfamily, and the subfamily placement of the donkey gene is the same as that of the characterised human protein, so the gene-level transfer is sound. Caveat for this specific gene model: A0A9L0K2P4 lacks the segment equivalent to human 288-319 (as in human isoform 2), which contains human Ser306, Lys317 and Trp319, the positions that PMID:23212377 maps to the NAD+-binding Asp/Thr/Tyr residues of bacterial NAD kinase; the GGDG motif is retained. Whether this splice form is active is untested. The same donkey gene also produces A0A8C4M4D0, which aligns gap-free to the full human canonical sequence and retains all three positions (file:EQUAS/NADK2/NADK2-bioinformatics/RESULTS-rereview.md). The annotation is accepted as a statement about the NADK2 gene product, with the isoform caveat recorded in suggested_questions.
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/NADK2-uniprot.txt
PANTHER; PTHR13158:SF5; NAD KINASE 2, MITOCHONDRIAL; 1.
PMID:23212377
although the GGDG motif and NE/D short motif were completely conserved, the Asp, Thr and Tyr residues were not, corresponding instead to Ser-306, Lys-317 and Trp-319
file:EQUAS/NADK2/NADK2-bioinformatics/RESULTS-rereview.md
The benchmark product therefore lacks part of the predicted NAD+-binding region; whether it is catalytically active is untested
GO:0005739 mitochondrion
IEA
GO_REF:0000120 		ACCEPT
Summary: Mitochondrion. Human NADK2 localises to mitochondria in HEK293A cells 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), and loss of NADK2 in patient fibroblasts lowers mitochondrial NADP(H) (PMID:24847004).
Reason: Experimentally established for the human ortholog in the same PANTHER subfamily and propagated by PAINT. The region equivalent to the human transit peptide (human 1-62) is conserved in the donkey sequence (donkey 97-158) but lies 96 residues from the N-terminus in all equid gene models, so the start site of the donkey protein is uncertain; the localisation itself is not in doubt at the gene level. Mitochondrial matrix (below) is the more specific location and is also accepted; this broader row agrees with it.
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
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/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 without transmembrane segments that is imported by an N-terminal targeting sequence and supplies NADP(H) to matrix NADPH-dependent enzymes such as dienoyl-CoA reductase and the first enzyme of lysine degradation (PMID:24847004).
Reason: The matrix is the specific compartment in which NADK2 acts; the enzymes whose deficiency results from NADK2 loss are matrix oxidoreductases. This is the most specific supported location.
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
PMID:24847004
DECR and also the first step in lysine degradation are performed by NADP-dependent oxidoreductases explaining their in vivo deficiency
GO:0019674 NAD+ metabolic process
IEA
GO_REF:0000118 		MODIFY
Summary: NAD+ metabolic process from TreeGrafter (PANTHER node PTN001407776), matching the PAINT IBD for this term at PTN004406956 seeded by human NADK2. The enzyme consumes NAD+ to make NADP+.
Reason: True but general. The step NADK2 itself catalyses is the phosphorylation of NAD+ that forms NADP+, and NAD kinase is the only NADP+ biosynthetic enzyme, so NADP+ biosynthetic process (GO:0006741), an is_a descendant of NAD+ metabolic process (checked in OLS), is the precise term. The participation test is met because NADK2 performs the step. Replaces the separate NEW row of the earlier version of this review.
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

Core Functions

Mitochondrial NAD(+) kinase that phosphorylates NAD(+) with ATP to form NADP(+), supplying the mitochondrial matrix NADP(H) pool.

Molecular Function:
NAD+ kinase activity
Directly Involved In:
NADP+ biosynthetic process
Cellular Locations:
mitochondrial matrix
Supporting Evidence:
  • PMID:23212377
    showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors
  • PMID:23212377
    confirming that C5orf33 protein is a mitochondrial NADK, and that its N-terminal 62 residues contain a functional mitochondrial-targeting sequence
  • 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/NADK2-uniprot.txt
    PANTHER; PTHR13158:SF5; NAD KINASE 2, MITOCHONDRIAL; 1.

References

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Suggested Questions for Experts

Q: Is the donkey NADK2 splice form represented by A0A9L0K2P4 (lacking the segment equivalent to human 288-319, which carries the positions mapped to the NAD+-binding site) expressed as protein, and is it catalytically active or a non-functional or regulatory variant?

Q: Does donkey NADK2 translation start at Met97 (equivalent to human Met1), placing the mitochondrial targeting sequence at the N-terminus, or at the upstream start used by the Ensembl equid gene models?

Suggested Experiments

Experiment: Clone donkey NADK2 transcripts from liver cDNA to define the start codon and the relative abundance of the full-length (A0A8C4M4D0-like) and 288-319-skipping (A0A9L0K2P4-like) splice forms, then assay the recombinant mature proteins for NAD kinase activity with ATP.

External Prediction Reviews

These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.

ProtNLM2 External predictions

View prediction review YAML · NADK2-protnlm-predictions-review.yaml · Review status: COMPLETE

Assessment of 3 ProtNLM2 GO predictions for donkey NADK2 (A0A9L0K2P4), scored for this exact sequence. A0A9L0K2P4 is the isoform-2-like splice product of the donkey NADK2 gene: it lacks the segment equivalent to human 288-319, which carries the TGSxAW NAD+-binding motif (W319) and the second-monomer active-site residue S306, while it keeps the GGDG motif, the ATP-contacting residues (human K76, S188, D161) and the extension-3/EMKA dimerization element (human 325-367). The full-length canonical-equivalent product of the same gene is A0A8C4M4D0. Predictions are judged against donkey GOA on any NADK2 product (NAD+ kinase activity, mitochondrion, mitochondrial matrix, NAD+ metabolic process) and the human ortholog Q4G0N4 (PMID:23212377, PMID:35868311, PMID:35733246). ATP binding and protein homodimerization are retained by this product and absent from donkey GOA (COR); phosphorylation, which here means phosphorylation of NAD+, is uncertain for this product because it lacks the NAD+-binding segment but keeps the ATP site and dimerization element, and its activity is untested (UNC). Non-GO ProtNLM2 outputs, not scored: subcellular location 'Mitochondrion' and function text 'Mitochondrial NAD(+) kinase that phosphorylates NAD(+) to yield NADP(+). Can use both ATP or inorganic polyphosphate as the phosphoryl donor', which reproduces the human Swiss-Prot function statement.

Source documents: projects/PROTNLM_EVALUATION/donkey40/donkey40-predictions.tsv · genes/EQUAS/NADK2/NADK2-goa.tsv · genes/human/NADK2/NADK2-uniprot.txt · genes/EQUAS/NADK2/NADK2-bioinformatics/RESULTS.md · genes/EQUAS/NADK2/NADK2-bioinformatics/RESULTS-rereview.md

Review score: 2 = concordant with evidence; 1 = uncertain; 0 = discordant with evidence. This is an assessment score, not a model probability.

GO:0042803 protein homodimerization activity GO_MF
COR — Correct novel prediction Review score: 2/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: Not in donkey GOA, nor on the sibling donkey product A0A8C4M4D0. Human NADK2 (same PANTHER subfamily PTHR13158:SF5) is a homodimer by crystal structure, SAXS and SEC-MALS (PMID:35868311), and an independent structure shows the same symmetrical homodimer (PMID:35733246). The element that mediates dimerization is extension 3/EMKA (human 325-367); deleting it gives a monomer. This accession retains that element: donkey 384-506 aligns to human 320-442. The segment it lacks (human 288-319) contributes S306 and A288 to the composite active site but is not the identified dimerization interface, so the product keeps the feature the prediction needs and the transfer stands.
Supporting Evidence:
GO:0005524 ATP binding GO_MF
COR — Correct novel prediction Review score: 2/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: Human NADK2 uses ATP as the phosphoryl donor (PMID:23212377). In the human structure the ATP site is formed by K76 next to the ATP triphosphate, S188 stabilising its phosphates, and the GGDG catalytic motif (PMID:35868311). All of these lie in human 1-287, which aligns to donkey 97-383 of this accession, and the GGDG motif is conserved (donkey 255-258). The segment this product lacks (human 288-319) contacts NAD+, not ATP, so the product keeps the ATP-binding features. ATP binding is not in donkey GOA, and NAD+ kinase activity is not a GO descendant of ATP binding, so this is a novel correct term rather than LSP.
Supporting Evidence:
  • PMID:23212377: "showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors"
  • PMID:35868311: "K76 is located in the vicinity of the metal ion (7.4 Å) and is predicted to lie next to the triphosphate moiety of ATP"
  • PMID:35868311: "as well as the S188, which appears to be readily available to stabilize the three phosphate groups of the phosphodonor ATP"
  • file:EQUAS/NADK2/NADK2-bioinformatics/RESULTS.md: "97-383 : 1-287"
GO:0016310 phosphorylation GO_BP
UNC — Uncertain Review score: 1/2
Prediction method: ProtNLM2 · Version: UniProt API snapshot 2026-10-14
Review rationale: For NADK2 this process is the phosphorylation of NAD+ to NADP+ (PMID:23212377). This accession provably lacks the segment equivalent to human 288-319: the donkey-human alignment aligns 0 of its 32 residues. The segment carries the TGSxAW NADK motif (human 314-319), whose W319 stacks the nicotinamide ring, and S306, which the second monomer contributes to the nicotinamide contacts (PMID:35868311). The product cannot form a complete NAD+ site of its own. The requirement of this segment for catalysis is inferred from the crystal structures; no mutagenesis of W319, S306 or the 288-319 segment and no activity assay of the isoform-2-equivalent product exist. The product keeps the GGDG catalytic motif, the ATP-site residues K76 and S188, the EMKA dimerization element and E379, which also contacts the nicotinamide in trans as E379-prime. It could therefore heterodimerize with the full-length product A0A8C4M4D0 and contribute residues to that partner's composite active site. This contribution is neither shown nor excluded. There is no positive refutation, and the missing segment does not fully account for the mechanism, so the call is uncertain rather than incorrect.
Supporting Evidence:

Deep Research

Cyberian

(NADK2-deep-research-cyberian.md)

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📚 Additional Documentation

Notes

(NADK2-notes.md)

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Bioinformatics Results

(RESULTS.md)

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📄 View Raw YAML

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