NADK2 (NAD kinase 2, mitochondrial; also known as C5orf33, MNADK, NADKD1) is the mitochondrial NAD(+) kinase (EC 2.7.1.23). Following import into the mitochondrial matrix and cleavage of its N-terminal transit peptide, the mature protein assembles into a homodimer that phosphorylates NAD(+) using ATP (and, in vitro, inorganic polyphosphate) to produce NADP(+). It is the source of the mitochondrial NADP(H) pool, the counterpart of the cytosolic NADK. The NADPH generated from this NADP(+) supplies NADP-dependent mitochondrial oxidoreductases, including 2,4-dienoyl-CoA reductase (DECR1) in polyunsaturated fatty acid beta-oxidation, the reductive step of lysine degradation, delta-1-pyrroline-5-carboxylate synthase (ALDH18A1/P5CS) in proline biosynthesis, mitochondrial one-carbon metabolism, and antioxidant defense against reactive oxygen species. The enzyme has low Km for NAD(+), only weak NADH kinase activity, and is feedback-inhibited by NADH, NADPH and NADP(+). It is broadly expressed with highest levels in liver. Loss-of-function mutations cause 2,4-dienoyl-CoA reductase deficiency (DECRD) / NADK2 deficiency, a mitochondrial NADP(H)-deficiency disorder presenting with severe infantile encephalopathy, lactic acidosis and hyperlysinemia.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003951 NAD+ kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of NAD+ kinase activity, the defining and experimentally established catalytic function of NADK2. This is the core molecular function of the gene. Reason: The IBA call matches the direct experimental characterization (IDA, PMID:23212377); NADK2 is the mitochondrial NAD(+) kinase (EC 2.7.1.23). 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: Phylogenetic (IBA) localization to mitochondrion, consistent with the experimentally confirmed mitochondrial localization of NADK2. A more precise compartment (mitochondrial matrix) is captured by other annotations. Reason: NADK2 carries an N-terminal mitochondrial transit peptide and localizes to mitochondria in human cells (IDA, PMID:23212377); the matrix is the specific submitochondrial 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 | ACCEPT | Summary: Phylogenetic (IBA) involvement in NAD+ metabolic process. NADK2 consumes NAD+ to make NADP+, so it participates in NAD+ metabolism; the more informative biological process is NADP+ biosynthesis. Reason: Consistent with the enzyme's reaction (NAD+ + ATP -> ADP + NADP+ + H+). NADP+ biosynthetic process (GO:0006741) is proposed as a more specific term. Supporting Evidence: 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 (ARBA/RHEA/EC) assignment of NAD+ kinase activity, mapped from EC 2.7.1.23 and RHEA:18629. Correct and redundant with the experimental IDA/IBA calls. Reason: The EC/RHEA mapping matches the UniProt catalytic activity and 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 UniProt Subcellular Location keyword mapping (SL-0173) to mitochondrion. Consistent with experimental localization. Reason: Matches the experimentally determined mitochondrial localization; the matrix is the specific compartment. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (ARBA) assignment to the mitochondrial matrix. NADK2 is a soluble matrix enzyme (cleaved transit peptide, no transmembrane segments), so the matrix is the correct and most informative compartment. Reason: The matrix localization is consistent with the transit-peptide-directed import of a soluble kinase and with Reactome/UniProt matrix placement. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion |
| GO:0019674 NAD+ metabolic process | IEA GO_REF:0000002 | ACCEPT | Summary: Electronic (InterPro IPR017437, ATP-NAD kinase PpnK C-domain) assignment to NAD+ metabolic process. Correct for an NAD kinase family member. Reason: The NAD kinase domain signature correctly implies participation in NAD+ metabolism; NADP+ biosynthesis (GO:0006741) is the more precise process. Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP |
| GO:0005739 mitochondrion | IDA GO_REF:0000052 | ACCEPT | Summary: Immunofluorescence (HPA) localization to mitochondrion. Corroborates the experimental mitochondrial localization. Reason: Independent IDA localization consistent with PMID:23212377 and the transit peptide. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: High-throughput mitochondrial proteomics places NADK2 in the high-confidence human mitochondrial proteome. Supports, but is less specific than, the matrix annotation. Reason: NADK2 is a bona fide component of the mitochondrial proteome; consistent with all other localization evidence. |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-8955030 | ACCEPT | Summary: Reactome TAS localization to the mitochondrial matrix, from the reaction "NADK2 dimer phosphorylates NAD+ to NADP+". This is the correct compartment for the NADK2 catalytic activity and is the core localization. Reason: The matrix is where the soluble NADK2 kinase acts; this Reactome event describes NADK2's own reaction. 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 | KEEP AS NON CORE | Summary: Reactome TAS matrix localization derived from an event in which NADK2 is a substrate of the LONP1 matrix protease ("LONP1 degrades mitochondrial matrix proteins"). The compartment (matrix) is correct, but this event describes LONP1's function, not NADK2's, so it is a non-core, indirect localization annotation. Reason: The matrix localization is accurate and consistent with the direct annotations, but this Reactome entry is about LONP1-mediated degradation of matrix proteins rather than NADK2's own biology. Supporting Evidence: Reactome:R-HSA-9838081 LONP1 degrades several mitochondrial matrix proteins |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9838093 | KEEP AS NON CORE | Summary: Reactome TAS matrix localization from an event in which NADK2 is bound as a substrate by the LONP1 matrix protease ("LONP1 binds mitochondrial matrix proteins"). Correct compartment but an indirect, non-core localization annotation. Reason: Matrix localization is accurate; the source event describes LONP1 substrate binding rather than NADK2's own function. 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: Direct experimental demonstration that purified NADK2 phosphorylates NAD+ to NADP+ using ATP (or inorganic polyphosphate), with defined kinetics. This is the defining core molecular function of NADK2. Reason: Purified recombinant protein forms NADP+ from NAD+ and ADP from ATP (TLC), with Km(NAD+) 0.022 mM; unambiguous NAD+ kinase (EC 2.7.1.23) 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 |
| GO:0005739 mitochondrion | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | ACCEPT | Summary: Direct experimental demonstration of mitochondrial localization by immunofluorescence and subcellular fractionation in human cells; transit-peptide dependent. Core localization (matrix compartment). Reason: FLAG-tagged full-length NADK2 localizes to mitochondria while the transit-peptide deletion does not; confirmed by mitochondrial/cytosolic fractionation. 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... | ACCEPT | Summary: Direct evidence that NADK2 activity contributes to mitochondrial NAD(P) metabolism (knockdown reduces mitochondrial NADK activity and perturbs ROS/redox balance). Core biological process; the anabolic product side is NADP+ biosynthesis. Reason: NADK2 catalyzes NAD+ -> NADP+ and its knockdown lowers mitochondrial NADK activity to 47% and elevates ROS, confirming a role in NAD(P) metabolism. Supporting Evidence: PMID:23212377 NADK activity of the mitochondrial fraction was decreased to 47% upon knockdown of C5orf33 |
| GO:0042803 protein homodimerization activity | IDA PMID:23212377 Identification and characterization of a human mitochondrial... | KEEP AS NON CORE | Summary: NADK2 functions as a homodimer (UniProt SUBUNIT; Reactome "NADK2 dimer"). This is a genuine oligomerization property but describes the enzyme's quaternary structure rather than its catalytic activity, so it is a non-core molecular function. Reason: The homodimer state is real and relevant to how the active enzyme assembles, but self-association is not the gene's core functional contribution (NAD+ kinase activity is); recorded as non-core. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt SUBUNIT: Homodimer. |
| GO:0006741 NADP+ biosynthetic process | IDA | NEW | Summary: Proposed new biological-process annotation. GOA records only the broader NAD+ metabolic process for NADK2, but the enzyme's anabolic product is NADP+; NADK2 is the source of mitochondrial NADP(+). UniProt already carries this term via InterPro (GO:0006741; P:NADP+ biosynthetic process; IEA:InterPro). Proposing it as the more specific, informative process term. Supporting Evidence: PMID:23212377 we detected the formation of NADP+ from NAD+ and of ADP from ATP file:human/NADK2/NADK2-uniprot.txt GO:0006741; P:NADP+ biosynthetic process; IEA:InterPro |
| GO:0005524 ATP binding | IDA | NEW | Summary: Proposed new molecular-function annotation. NADK2 binds ATP as the phosphoryl donor of the NAD kinase reaction (Km for ATP 1.7 mM); ATP-binding is a UniProt keyword (ATP-binding) and is intrinsic to the catalytic mechanism but is not present in the GOA experimental set. Supporting Evidence: file:human/NADK2/NADK2-uniprot.txt Reaction=NAD(+) + ATP = ADP + NADP(+) + H(+); Xref=Rhea:RHEA:18629 |
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