NADK2 (NAD kinase 2, mitochondrial; also known as C5orf33, MNADK, NADKD1) is the mitochondrial NAD(+) kinase (EC 2.7.1.23). It is imported into mitochondria by an N-terminal transit peptide and acts as a soluble enzyme that phosphorylates NAD(+) using ATP (and, in vitro, inorganic polyphosphate) to produce NADP(+). It is the source of the mitochondrial NADP(H) pool, separate from the cytosolic pool made by NADK. The enzyme has a low Km for NAD(+), only weak NADH kinase activity, and is inhibited by NADH, NADPH and NADP(+). Crystal structures show a constitutively active homodimer, held together by an insert that is characteristic of animal mitochondrial NAD kinases and that prevents the tetramer formed by other NAD kinases; acetylation of lysines near the active site inhibits it. Mitochondrial NADPH derived from NADK2 is used by NADPH-dependent matrix enzymes, including 2,4-dienoyl-CoA reductase (DECR1) in polyunsaturated fatty acid oxidation, the first step of lysine degradation, and delta-1-pyrroline-5-carboxylate synthase (P5CS) in proline biosynthesis, which makes NADK2-deficient cells proline auxotrophs. NADK2 is broadly expressed, with high levels in liver; Nadk2 knockout mice show stress-induced hepatic steatosis. Biallelic loss-of-function variants cause NADK2 deficiency (2,4-dienoyl-CoA reductase deficiency, DECRD), a mitochondrial NADP(H) deficiency with infantile-onset encephalopathy, elevated C10:2-carnitine and hyperlysinemia.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003951 NAD+ kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT transfer of NAD+ kinase activity from PTN004406956, seeded by human NADK2 and mouse Nadk2. NAD+ kinase activity is the defining, experimentally established activity of NADK2. Reason: The IBA agrees with the direct biochemical evidence (IDA, PMID:23212377). Human NADK2 appearing among the seeds is expected, because its own IDA is one of the descendant evidences behind the IBD. The family review (interpro/panther/PTHR13158) judges the node too deep, because it is the horizontal-transfer root and passes the term to an uncharacterised bacterial branch. Human NADK2 is inside the eukaryotic clade where the activity is established, so this row is unaffected. Supporting Evidence: PMID:23212377 showing that C5orf33 protein has NADK activity using ATP and poly(P) as phosphoryl donors |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT transfer of mitochondrial localisation from the eukaryotic node PTN000994636, seeded by human and mouse NADK2. The organelle claim is directly established for human NADK2. Reason: NADK2 has an N-terminal mitochondrial transit peptide and localises to mitochondria by imaging and fractionation (IDA, PMID:23212377). The IBA stops at the organelle because its seeds were assayed at that level; the matrix rows record the sub-compartment. 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:0019674 NAD+ metabolic process | IBA GO_REF:0000033 | MODIFY | Summary: PAINT transfer of NAD+ metabolic process from PTN004406956. True, because NADK2 consumes NAD+, but the process NADK2 itself carries out is the synthesis of NADP+. Reason: The term is correct but general. NADP+ biosynthetic process (GO:0006741) is an is_a descendant of NAD+ metabolic process (checked in QuickGO) and names the step NADK2 catalyses, NAD+ + ATP -> NADP+ + ADP. NAD kinase is the only enzyme that makes NADP+, and NADK2 is the mitochondrial one. GO:0006741 is already on the UniProt entry through InterPro. Same treatment as the horse and donkey NADK2 reviews and the PTHR13158 family review. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN004406956 SUPPORTS TRANSFER Family root node; human NADK2 is in the eukaryotic clade below it, where the function is established. 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 NAD kinase is the sole NADP(+) biosynthetic enzyme PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP |
| GO:0003951 NAD+ kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of NAD+ kinase activity (EC 2.7.1.23, RHEA:18629). Same claim as the IBA and IDA rows. Reason: The EC/Rhea mapping matches the UniProt catalytic activity, which is tied to the direct experimental evidence. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt Reaction=NAD(+) + ATP = ADP + NADP(+) + H(+); Xref=Rhea:RHEA:18629 |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic annotation from the UniProt subcellular location (SL-0173, Mitochondrion). The UniProt location is itself tied to PMID:23212377. Reason: Matches the experimentally determined mitochondrial localisation. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:23212377}. |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (ARBA) assignment to the mitochondrial matrix. NADK2 is a soluble enzyme with a cleavable transit peptide and no transmembrane segment, and the enzymes that depend on its product are matrix enzymes. Reason: Two independent lines place NADK2 in the matrix. It is imported by an N-terminal targeting sequence and has no membrane anchor, and its product is required by matrix NADPH-dependent enzymes (DECR1, the first step of lysine degradation, P5CS). Reactome places the NADK2 reaction in the matrix. Submitochondrial fractionation has not been reported, so the matrix is inferred rather than measured. Supporting Evidence: Reactome:R-HSA-8955030 Mitochondrial NAD kinase 2 (NADK2 aka C5orf33, MNADK, NADKD1) uses ATP to phosphorylate NAD+ to NADP+ 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:0000002 | MODIFY | Summary: InterPro2GO assignment (IPR017437, ATP-NAD kinase PpnK-type C-terminal domain) to NAD+ metabolic process. 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 NAD kinase is the sole NADP(+) biosynthetic enzyme file:human/NADK2/NADK2-uniprot.txt InterPro; IPR017437; ATP-NAD_kinase_PpnK-typ_C. |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: Human Protein Atlas immunofluorescence localisation to mitochondria. Independently corroborates PMID:23212377. Reason: Whole-organelle imaging that cannot resolve the sub-compartment; the organelle claim is correct and central to NADK2 function. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-confidence human mitochondrial proteome. Organelle proteomics places NADK2 in mitochondria. Reason: Organelle proteomics cannot resolve the sub-compartment; the organelle claim agrees with all other localisation evidence. Supporting Evidence: PMID:34800366 it is crucial to establish a high-confidence inventory of the human mitochondrial proteome |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-8955030 | ACCEPT | Summary: Reactome places its reaction "NADK2 dimer phosphorylates NAD+ to NADP+" in the mitochondrial matrix. This is NADK2's own catalytic event and the compartment where it acts. Reason: Consistent with the import of a soluble kinase by a transit peptide and with the matrix location of the NADPH-dependent enzymes it supplies. Supporting Evidence: Reactome:R-HSA-8955030 Mitochondrial NAD kinase 2 (NADK2 aka C5orf33, MNADK, NADKD1) uses ATP to phosphorylate NAD+ to NADP+ |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9838081 | ACCEPT | Summary: Reactome matrix location from the event "LONP1 degrades mitochondrial matrix proteins", in which NADK2 is a LONP1 substrate. The event is about LONP1, but the location it records for NADK2 is the same matrix claim as the other rows. Reason: The review judges the claim, not the source event. NADK2 being a matrix protein is supported by the other rows, and listing it as a LONP1 substrate is consistent with that. This row adds no information about NADK2 function beyond the location. Supporting Evidence: Reactome:R-HSA-9838081 LONP1 degrades several mitochondrial matrix proteins |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9838093 | ACCEPT | Summary: Reactome matrix location from the event "LONP1 binds mitochondrial matrix proteins", in which NADK2 is a LONP1 substrate. Same matrix claim as the other rows. Reason: As for the other LONP1 event, the location is correct and consistent with the rows that rest on NADK2's own biology. Supporting Evidence: Reactome:R-HSA-9838093 LONP1 binds substrate proteins and degrades them using an ATP-dependent mechanism |
| GO:0003951 NAD+ kinase activity | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | ACCEPT | Summary: Purified recombinant NADK2 phosphorylates NAD+ to NADP+ using ATP or inorganic polyphosphate, with defined kinetics. This is the defining core activity of NADK2. Reason: The purified protein forms NADP+ from NAD+ and ADP from ATP (Km for NAD+ 0.022 mM). Its NADH kinase activity is only about 10% of the NAD+ kinase activity, so NAD+ kinase, not NADH kinase, is the activity to annotate. An independent study of recombinant human NADK2 (as MNADK) also measured NAD kinase activity. Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP (Fig. 2c), showing that C5orf33 protein has NADK activity PMID:23212377 the NADHK activity of C5orf33 protein was only 10% of its NADK activity PMID:23616928 a recombinant MNADK has NAD kinase activity |
| GO:0005739 mitochondrion | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | ACCEPT | Summary: Immunofluorescence and fractionation in HEK293A cells place NADK2 in mitochondria, and the localisation depends on the N-terminal 62 residues. Reason: FLAG-tagged full-length NADK2 localises to mitochondria while the transit-peptide deletion does not; fractionation agrees. Whole-organelle assays, so the organelle term is the right level for this row. Supporting Evidence: PMID:23212377 C5orf33 protein was localized in the mitochondrial fraction but not in the cytosolic fraction |
| GO:0019674 NAD+ metabolic process | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | MODIFY | Summary: Direct evidence that NADK2 converts NAD+ to NADP+, and that knockdown lowers mitochondrial NAD kinase activity. True, but the process NADK2 carries out is NADP+ synthesis. Reason: The experiment supports the more specific process. NADK2 catalyses the NAD+ to NADP+ step, and its knockdown lowers mitochondrial NADK activity to 47%; NADK2 loss in patient fibroblasts lowers mitochondrial NADP(H). NADP+ biosynthetic process (GO:0006741) is the is_a descendant that names this step. Proposed replacements: NADP+ biosynthetic process Supporting Evidence: PMID:23212377 NADK activity of the mitochondrial fraction was decreased to 47% upon knockdown of C5orf33 PMID:24847004 NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts |
| GO:0042803 protein homodimerization activity | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | KEEP AS NON CORE | Summary: NADK2 is a homodimer. The original statement is a curator inference from PMID:23212377 (UniProt ECO:0000305), but two later crystal structures confirm a dimer, held together by an insert specific to animal mitochondrial NAD kinases. Reason: The homodimer is now well supported and is required for activity, but self-association describes how the enzyme is assembled rather than what it contributes; NAD+ kinase activity is the core function. Same action as the horse NADK2 review. Supporting Evidence: 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: Should NADK2 carry a proline biosynthesis or lysine catabolism process term? This review does not add one. NADK2 supplies the NADPH that P5CS and the lysine-degradation oxidoreductase use, but performs no step of either pathway, so by the participation test it is a supplier of a cofactor rather than a participant. Would a has_input/has_part relation in a GO-CAM model (NADK2 -> NADP+ -> P5CS) express this better?
Q: Is isoform 2 (Q4G0N4-2), which lacks residues 288-319 including the NAD+-stacking Trp319, catalytically active, and is it expressed as protein?
Q: Does the weak NADH kinase activity of NADK2 (about 10% of its NAD+ kinase activity in vitro) contribute to mitochondrial NADPH in vivo?
Experiment: Express the mature forms of NADK2 isoforms 1 and 2 and assay NAD+ kinase activity and dimerisation; quantify isoform 2 protein in liver mitochondria by targeted proteomics.
Type: enzyme assay and targeted proteomics
Experiment: Confirm the matrix location of NADK2 by protease protection of isolated mitochondria and mitoplasts, with matrix and intermembrane-space markers.
Type: submitochondrial fractionation
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