NADK2 is the mitochondrial NAD(+) kinase (EC 2.7.1.23) of the horse, the ortholog of human and mouse NADK2 within the PANTHER mitochondrial NAD kinase subfamily (PTHR13158:SF5). It is a soluble matrix enzyme with an N-terminal mitochondrial targeting region and a C-terminal NAD kinase catalytic domain carrying the conserved GGDG and NE/D motifs. By homology with the experimentally characterised human enzyme, it phosphorylates NAD(+) with ATP (or inorganic polyphosphate in vitro) to form NADP(+), and so supplies the mitochondrial NADP(H) pool used by NADPH-dependent matrix enzymes such as 2,4-dienoyl-CoA reductase in polyunsaturated fatty acid oxidation, the first step of lysine degradation, proline synthesis and antioxidant defence. The human enzyme is thought to act as a homodimer. The UniProt entry A0A3Q2GYY5 is one of several horse NADK2 gene models; it has a 96-residue N-terminal extension that the human and mouse proteins lack and a 22-residue alternative exon shared with canonical mouse NADK2.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003951 NAD+ kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT transfer of NAD+ kinase activity from node PTN004406956, seeded by experimental annotations on human NADK2 (Q4G0N4) and mouse Nadk2. The horse protein sits in the same PANTHER subfamily (PTHR13158:SF5) and retains every catalytic motif identified in human NADK2. Reason: Purified human NADK2 forms NADP+ from NAD+ and ATP; horse NADK2 is a subfamily member, 96.8% identical over the human-aligned region, with the GGDG and NE/D motifs and the NADK2-specific Ser/Lys/Trp NAD-site residues conserved. This is the core activity. Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP file:HORSE/NADK2/NADK2-uniprot.txt PANTHER; PTHR13158:SF5; NAD KINASE 2, MITOCHONDRIAL; 1. file:HORSE/NADK2/NADK2-bioinformatics/RESULTS.md Catalytic motifs are present |
| GO:0003951 NAD+ kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined electronic assignment of NAD+ kinase activity (EC 2.7.1.23, ortholog transfer from human and mouse NADK2). Agrees with the IBA row and the sequence evidence. Reason: Same claim as the IBA row, grounded by subfamily membership and conserved catalytic motifs; the orthologs it draws on have direct biochemical evidence. Supporting Evidence: PMID:23212377 showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors file:HORSE/NADK2/NADK2-uniprot.txt PANTHER; PTHR13158:SF5; NAD KINASE 2, MITOCHONDRIAL; 1. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT transfer of mitochondrial localisation from node PTN000994636 (seeded by human and mouse NADK2). Consistent with the conserved NADK2 targeting region. Reason: Human NADK2 is mitochondrial by fractionation and imaging, and the localisation depends on its N-terminal transit peptide; horse NADK2 is in the same subfamily and keeps the human-type N-terminal region from its residue 97. Mitochondrion is what the direct experiments show; the matrix row below records the inferred sub-compartment. Supporting Evidence: PMID:23212377 C5orf33 protein was localized in the mitochondrial fraction but not in the cytosolic fraction |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic transfer of mitochondrial localisation from human NADK2. Same claim as the IBA row. Reason: Consistent with the experimental localisation of the human ortholog and the subfamily placement. Supporting Evidence: PMID:23212377 the C5orf33 protein is localized in mitochondria, whereas Ξ62C5orf33 protein is not, confirming that C5orf33 protein is a mitochondrial NADK |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment to the mitochondrial matrix. NADK2 is a soluble enzyme with a cleavable transit peptide and no membrane segments, and it supplies matrix NADP(H). Reason: The matrix is where the enzyme acts. The human NADK2 reaction is placed in the matrix by Reactome, and loss of NADK2 depletes mitochondrial NADP(H) and blocks matrix NADPH-dependent enzymes such as DECR. Supporting Evidence: PMID:24847004 NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts Reactome:R-HSA-8955030 Mitochondrial NAD kinase 2 (NADK2 aka C5orf33, MNADK, NADKD1) uses ATP to phosphorylate NAD+ to NADP+ |
| GO:0019674 NAD+ metabolic process | IBA GO_REF:0000033 | MODIFY | Summary: PAINT transfer of NAD+ metabolic process from PTN004406956. Correct, because the enzyme consumes NAD+, but the specific process it performs is the synthesis of NADP+. Reason: The term is true but general. NADP+ biosynthetic process (GO:0006741) is an is_a descendant of NAD+ metabolic process and names the step NADK2 itself catalyses (NAD+ + ATP -> NADP+ + ADP). The replacement refines the PAINT term and does not contradict it. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN004406956 SUPPORTS TRANSFER Node in PTHR13158 containing subfamily SF5; horse NADK2 descends from it. UniProtKB:Q4G0N4 Β· human NADK2 SUPPORTS TRANSFER Direct biochemical evidence of NAD+ to NADP+ conversion (PMID:23212377). Proposed replacements: NADP+ biosynthetic process Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP PMID:24847004 NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis |
| GO:0019674 NAD+ metabolic process | IEA GO_REF:0000120 | MODIFY | Summary: Electronic assignment of NAD+ metabolic process (InterPro IPR017437 and ortholog transfer). Same claim as the IBA row. Reason: As for the IBA row, NADP+ biosynthetic process (GO:0006741) is the specific descendant term for the reaction NADK2 catalyses. Proposed replacements: NADP+ biosynthetic process Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP |
| GO:0042803 protein homodimerization activity | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: Ensembl Compara transfer of homodimerization from human NADK2. The UniProt homodimer statement for human NADK2 is a curator inference from PMID:23212377, but two later crystal structures of human NADK2 show a dimer. PAINT did not propagate this term. Reason: Human NADK2 is a structurally confirmed homodimer, and horse NADK2 is a near-identical ortholog in the same subfamily, so the transfer is sound. It describes quaternary structure rather than the enzyme's catalytic contribution, so it is kept as non-core, as in the human NADK2 review. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt SUBUNIT: Homodimer. PMID:35868311 We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. PMID:35733246 the absence of cooperativity indicates that NADK2 forms a constitutively active homodimer that does not dissociate |
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Download this section (compressed HTML)Q: Is the 96-residue N-terminal extension of the A0A3Q2GYY5 gene model (upstream of the human-equivalent start Met at residue 97) supported by horse transcript or proteomic evidence, or should the model start at Met-97 so the mitochondrial transit peptide is N-terminal?
Q: Do the horse NADK2 splice forms with and without the 22-residue alternative exon (A0A3Q2GYY5 versus F7DCV0) and the form lacking human residues 288-319 (A0A3Q2H586) differ in catalytic activity or mitochondrial targeting?
Experiment: Map the native N-terminus of horse liver NADK2 by N-terminal proteomics or 5' RACE, and test whether a Met-1 construct of A0A3Q2GYY5 is imported into mitochondria as efficiently as a Met-97 construct.
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