NADSYN1 (glutamine-dependent NAD(+) synthetase; EC 6.3.5.1) is a cytosolic homohexameric enzyme that catalyzes the final, committed step of nicotinamide adenine dinucleotide (NAD+) de novo biosynthesis. It performs the ATP-dependent amidation of deamido-NAD+ (nicotinate adenine dinucleotide, NaAD) to NAD+, using L-glutamine as the physiological nitrogen donor, which is hydrolyzed to L-glutamate. This same reaction is the last step of both the de novo (tryptophan/kynurenine) route and the Preiss-Handler route, which converge at deamido-NAD+. The protein is bifunctional, with an N-terminal carbon-nitrogen (CN) hydrolase / nitrilase-family glutaminase domain that hydrolyzes glutamine to supply ammonia in situ (catalytic nucleophile Cys-175) and a C-terminal NAD synthetase (ligase) domain that carries out the ATP-dependent amidation. NADSYN1 can also use free ammonia as an alternative amide donor, but its low KM for glutamine indicates glutamine is the physiological donor. Bi-allelic loss-of-function variants cause a congenital NAD deficiency disorder (Vertebral, cardiac, renal, and limb defects syndrome 3; VCRL3), an autosomal recessive, often lethal condition with cardiac, renal, vertebral, and limb malformations.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0034354 'de novo' NAD+ biosynthetic process from L-tryptophan | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) placement of NADSYN1 in the de novo NAD+ biosynthesis pathway from L-tryptophan. NADSYN1 catalyzes the final step of this pathway, so this is a core biological process; independently supported by experimental IMP evidence (PMID:31883644). Reason: NADSYN1 performs the last step (deamido-NAD+ to NAD+) of the de novo NAD+ route that begins with tryptophan-derived quinolinate; the phylogenetic call matches the experimentally established function. Supporting Evidence: file:human/NADSYN1/NADSYN1-uniprot.txt Catalyzes the final step of the nicotinamide adenine |
| GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) assignment of the core catalytic activity of NADSYN1, glutamine-hydrolyzing NAD+ synthase (EC 6.3.5.1). This is the defining molecular function of the gene and is independently supported by experimental evidence (EXP PMID:12547821, IMP PMID:31883644). Reason: This is the core molecular function; the IBA call is fully concordant with direct enzymatic characterization of human NADSYN1. Supporting Evidence: file:human/NADSYN1/NADSYN1-uniprot.txt NAD(+) synthase [glutamine-hydrolyzing] |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: Phylogenetic (IBA) cytoplasmic localization. NADSYN1 is a soluble cytosolic enzyme; "cytoplasm" is a correct but broad parent of the specific "cytosol" localization (GO:0005829) supported by Reactome TAS. Reason: The more precise and well-supported location is cytosol (GO:0005829); the generic cytoplasm term is uninformative relative to it. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: GRANULARITY MISMATCH |
| GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (IEA) assignment of the core NAD+ synthase (glutamine-hydrolyzing) activity via ARBA/InterPro/RHEA/EC mapping. Correct and redundant with the experimental annotations. Reason: Concordant with experimental (EXP/IMP) evidence for the core catalytic function; the InterPro/EC/RHEA mapping (EC:6.3.5.1, RHEA:24384) is exactly right. |
| GO:0004359 glutaminase activity | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Electronic (IEA/InterPro) assignment of glutaminase activity, derived from the N-terminal CN-hydrolase (nitrilase-family) domain that hydrolyzes L-glutamine to L-glutamate + NH3 to supply the nitrogen for NAD+ synthesis. This is a genuine partial reaction of the enzyme, but it is an accessory/supporting activity subordinate to the core NAD+ synthase activity, not an independent metabolic role. Reason: The glutaminase (CN-hydrolase) partial reaction is real and mechanistically essential (Cys-175 mutation abolishes glutamine-dependent NAD synthesis) but functions as the amide-donor-generating half-reaction of the glutamine-hydrolyzing NAD+ synthase; it is not a standalone glutaminase in physiology. Supporting Evidence: PMID:12547821 the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Electronic (IEA/InterPro) assignment of ATP binding, consistent with the C-terminal NAD synthetase (ligase) domain, which uses ATP to activate the NaAD carboxylate during amidation (ATP-binding P-loop at residues 355-362). A supporting molecular function underpinning the core catalytic activity. Reason: ATP binding is a real, required activity of the ligase domain but is a supporting feature of the catalytic mechanism rather than the gene's core function. Supporting Evidence: file:human/NADSYN1/NADSYN1-uniprot.txt ATP-binding |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Electronic (IEA/InterPro) cytoplasmic localization. Correct but broad; the specific cytosol localization (GO:0005829) is better supported. Reason: Redundant broad parent of cytosol; NADSYN1 is a soluble cytosolic enzyme. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: GRANULARITY MISMATCH |
| GO:0005829 cytosol | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (IEA/ARBA) cytosol localization, concordant with the Reactome TAS annotation and with the biology of NADSYN1 as a soluble cytosolic hexameric enzyme. This is the correct specific subcellular location. Reason: Cytosol is the precise, well-supported localization of NADSYN1. Supporting Evidence: Reactome:R-HSA-197271 Cytosolic hexameric NAD synthase 1 (NADSYN1) catalyses the final step in the biosynthesis of NAD+ |
| GO:0009435 NAD+ biosynthetic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Electronic (IEA) assignment to NAD+ biosynthetic process, the general parent process. NADSYN1 catalyzes the last step of NAD+ biosynthesis; correct but less specific than the de novo-from-tryptophan term (GO:0034354). Reason: Accurate but broad; the specific de novo NAD+ biosynthesis term better captures the pathway context, so this general parent is retained as non-core. Supporting Evidence: file:human/NADSYN1/NADSYN1-uniprot.txt Cofactor biosynthesis; NAD(+) biosynthesis |
| GO:0034354 'de novo' NAD+ biosynthetic process from L-tryptophan | IEA GO_REF:0000117 | ACCEPT | Summary: Electronic (IEA/ARBA) assignment to de novo NAD+ biosynthesis from L-tryptophan. Correct and redundant with the IBA and IMP annotations to the same term; this is a core biological process for NADSYN1. Reason: NADSYN1 performs the final step of the de novo (tryptophan/kynurenine) NAD+ pathway; concordant with experimental IMP evidence (PMID:31883644). Supporting Evidence: file:human/NADSYN1/NADSYN1-uniprot.txt Catalyzes the final step of the nicotinamide adenine |
| GO:0005515 protein binding | IPI PMID:25416956 A proteome-scale map of the human interactome network. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a high-throughput proteome-scale interactome map (Rolland et al. 2014, systematic Y2H). The single partner (Q9H614) has no established functional relationship to NADSYN1's catalytic role and the term is uninformative. Reason: Non-specific bare protein-binding term from a systematic interactome screen; no biological interpretation for NADSYN1 function. Retained (not removed) per policy on IPI protein-binding annotations. |
| GO:0005515 protein binding | IPI PMID:31515488 Extensive disruption of protein interactions by genetic vari... | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from a large-scale interactome study of variant effects on protein interactions (Fragoza et al. 2019). Partner Q9NTM9 (CUTC). Uninformative for the gene's molecular function. Reason: Non-specific bare protein-binding term from a high-throughput screen; no functional significance established for NADSYN1. Retained per IPI policy. |
| GO:0005515 protein binding | IPI PMID:32296183 A reference map of the human binary protein interactome. | MARK AS OVER ANNOTATED | Summary: Bare "protein binding" from the HuRI reference binary interactome map (Luck et al. 2020). Multiple partners (ANKS1A, COL8A1, CREB5, CUTC, FRS3, GPANK1, HGS, HOXC8, MYO15B, NOXA1, NTAQ1, TFG). None has an established functional link to NADSYN1's enzymatic role; the term is uninformative. Reason: Non-specific bare protein-binding term from a systematic binary interactome screen; no biological interpretation for NADSYN1 function. Retained per IPI policy. |
| GO:0034354 'de novo' NAD+ biosynthetic process from L-tryptophan | IMP PMID:31883644 Bi-allelic Mutations in NADSYN1 Cause Multiple Organ Defects... | ACCEPT | Summary: Experimental (IMP) evidence that NADSYN1 functions in de novo NAD+ biosynthesis from tryptophan. Bi-allelic loss-of-function variants cause congenital NAD deficiency disorder (VCRL3) with severely reduced NAD levels, demonstrated by yeast complementation and enzymatic assays. Core biological process. Reason: Directly demonstrates NADSYN1's essential role in the de novo NAD+ pathway in humans; loss of function reduces NAD levels and causes NAD-deficiency malformations. Supporting Evidence: PMID:31883644 NADSYN1, encoding NAD synthetase 1, the final enzyme of the PMID:31883644 show impaired enzymatic activity and severely reduced NAD levels |
| GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity | EXP PMID:12547821 Molecular identification of human glutamine- and ammonia-dep... | ACCEPT | Summary: Direct experimental (EXP) characterization of human NADSYN1 as a glutamine-hydrolyzing NAD+ synthetase. Hara et al. cloned and biochemically assayed the enzyme, showing it uses glutamine (and ammonia) as amide donor, that the CN-hydrolase domain confers glutamine dependency (Cys-175 mutation abolishes glutamine use), and determined kinetic parameters. This is the definitive evidence for the core molecular function. Reason: Gold-standard experimental evidence for the core catalytic activity of NADSYN1. Supporting Evidence: PMID:12547821 NADsyn1 uses glutamine as well as ammonia as an amide donor PMID:12547821 the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis |
| GO:0003952 NAD+ synthase (glutamine-hydrolyzing) activity | IMP PMID:31883644 Bi-allelic Mutations in NADSYN1 Cause Multiple Organ Defects... | ACCEPT | Summary: Experimental (IMP) evidence for the NAD+ synthase (glutamine-hydrolyzing) activity of NADSYN1 via disease-variant functional assays. Missense VCRL3 variants show decreased NAD(+) synthase (glutamine-hydrolyzing) activity and reduced NAD levels, confirming this is the enzyme's core activity in vivo. Reason: Independently corroborates the core catalytic function via loss-of-function variant analysis in patients. Supporting Evidence: PMID:31883644 show impaired enzymatic activity and severely reduced NAD levels |
| GO:0005829 cytosol | TAS Reactome:R-HSA-197271 | ACCEPT | Summary: Traceable author statement (Reactome) that NADSYN1 acts as a cytosolic hexamer catalyzing the final NAD+ step. This is the correct, specific subcellular localization of the enzyme. Reason: Cytosol is the well-established localization of NADSYN1 as a soluble cytosolic hexameric enzyme. Supporting Evidence: Reactome:R-HSA-197271 Cytosolic hexameric NAD synthase 1 (NADSYN1) catalyses the final step in the biosynthesis of NAD+ |
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