GO:0016646 oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0016646 describes a molecular function: oxidation-reduction reactions where a CH-NH group donates hydrogen/electrons and NAD+ or NADP+ is the acceptor.
• This activity is central to cellular redox balance and is tightly linked to NADPH/NADP+ and NADH/NAD+ pools.
• Key enzymes include NAD kinases (NADK, NADK2) that generate NADP(H), and dehydrogenases that use NAD(P)+ as electron acceptors.
• Mitochondrial NADP(H) generation by NADK2 is essential for proline biosynthesis and fatty acid synthesis/lipoylation.
• Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis, linking this activity to one-carbon metabolism.
• Dysregulation of NAD(P)-dependent redox reactions is implicated in metabolic disorders, obesity, and cancer.
Description
GO:0016646, oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor, is a molecular function term in the Gene Ontology that defines a specific class of redox reactions. In these reactions, a CH-NH group (a carbon-hydrogen bond adjacent to a nitrogen in an amine or imine) serves as the hydrogen or electron donor, while NAD+ or NADP+ acts as the electron acceptor, being reduced to NADH or NADPH, respectively. This activity is fundamental to cellular metabolism, as it directly couples the oxidation of organic substrates to the generation of reduced nicotinamide cofactors, which are essential for biosynthesis, antioxidant defense, and energy production. The importance of GO:0016646 lies in its role in maintaining cellular redox homeostasis. NADPH, produced by many of these reactions, is a critical reducing agent for anabolic pathways such as fatty acid synthesis, proline biosynthesis, and folate metabolism. NADH, on the other hand, feeds electrons into the mitochondrial electron transport chain for ATP production. Enzymes annotated with this activity include NAD kinases (NADK, NADK2) that phosphorylate NAD+ to NADP+, and various dehydrogenases that utilize NAD(P)+ as a cofactor. Researchers study GO:0016646 to understand how cells balance oxidative and reductive processes, how metabolic flux is directed toward biosynthesis or energy production, and how perturbations in these pathways contribute to diseases such as obesity, cancer, and metabolic disorders. The term provides a framework for annotating gene products and for designing experiments that probe redox biology at the molecular, cellular, and organismal levels.
oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor At A Glance
| GO ID | GO:0016646 |
|---|---|
| GO term | oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Catalyzes redox reactions where a CH-NH group donates electrons to NAD+ or NADP+ |
| EC number | 1.5.1.- (oxidoreductases acting on CH-NH group of donors with NAD or NADP as acceptor) |
| Cofactor | NAD+ or NADP+ as electron acceptor |
| Substrate donor | CH-NH group (e.g., in amino acids, amines, imines) |
| Related pathways | Proline biosynthesis, fatty acid synthesis, folate metabolism, redox homeostasis |
What Is GO:0016646?
GO:0016646 is defined by the Gene Ontology as the catalysis of an oxidation-reduction (redox) reaction in which a CH-NH group acts as a hydrogen or electron donor and reduces NAD or NADP. In simpler terms, it describes enzymes that transfer electrons from a carbon-nitrogen (CH-NH) group of a substrate to NAD+ or NADP+, converting them to NADH or NADPH. This activity is a subset of oxidoreductase functions and is specific to the electron acceptor (NAD or NADP) and the donor group (CH-NH).
Why Is oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor Important in Cell Biology?
GO:0016646 is critically important because it governs the production of NADH and NADPH, which are central to cellular energy metabolism and biosynthesis. NADPH is required for reductive biosynthesis (e.g., fatty acids, proline) and for maintaining the glutathione antioxidant system, while NADH is a key electron donor for oxidative phosphorylation. Disruption of these reactions can lead to metabolic imbalances, oxidative stress, and diseases such as obesity, cancer, and mitochondrial disorders.
• Maintains cellular redox balance by regenerating NAD+ and producing NADH/NADPH.
• Supports anabolic pathways like proline biosynthesis and fatty acid synthesis.
• Provides NADPH for folate-dependent nucleotide synthesis.
• Links to obesity and metabolic syndrome via nicotinamide N-methyltransferase (NNMT) and NAD+ salvage.
• Involved in mitochondrial metabolism and lipoylation of enzymes.
• Essential for photosynthesis in plants via NADP+ reduction.
• Regulates NADP(H) balance in chloroplasts through NADP(H) phosphatases.
• Potential target for cancer therapy due to altered NAD(P) metabolism in tumors.
• Plays a role in neuroprotection by maintaining redox homeostasis (implied by general redox biology).
• Key for understanding enzyme mechanisms and designing inhibitors.
What Happens During oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor?
Substrate Binding and CH-NH Group Activation
In simple terms: The enzyme grabs the substrate and positions the CH-NH group for electron removal.
The reaction begins with the binding of a substrate containing a CH-NH group (e.g., an amino acid or amine) to the enzyme's active site. The enzyme orients the substrate so that the carbon-hydrogen bond adjacent to the nitrogen is accessible for hydride transfer. This step often involves conformational changes that bring the substrate close to the NAD(P)+ cofactor.
Hydride Transfer to NAD(P)+
In simple terms: A hydride ion (H-) is moved from the substrate to NAD+ or NADP+, reducing it to NADH or NADPH.
The core catalytic event is the transfer of a hydride ion from the CH-NH group to the nicotinamide ring of NAD+ or NADP+. This reduces NAD(P)+ to NAD(P)H. The reaction is stereospecific and depends on the precise alignment of the substrate and cofactor. Enzymes like NAD kinases indirectly support this by generating NADP+ from NAD+.
Product Release and Cofactor Regeneration
In simple terms: The oxidized substrate and NADH/NADPH are released, and the enzyme is ready for another round.
After hydride transfer, the oxidized substrate (now containing a C=N double bond or imine) is released, along with NADH or NADPH. The reduced cofactor can then be used in biosynthetic reactions or reoxidized by the electron transport chain. The enzyme returns to its initial state to catalyze another reaction. In mitochondria, NADPH produced by this activity fuels proline biosynthesis and fatty acid synthesis.
Integration with Cellular Metabolism
In simple terms: The products of this reaction feed into other metabolic pathways.
NADH and NADPH generated by GO:0016646 are central to cellular metabolism. NADPH is used for reductive biosynthesis (e.g., fatty acids, proline) and antioxidant defense, while NADH is oxidized by the respiratory chain to produce ATP. In plants, NADPH is critical for the Calvin cycle and photosynthesis. In Arabidopsis, NADP(H) phosphatases regulate the balance of these cofactors.
Key Genes Involved in GO:0016646 oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor
The following genes encode enzymes that exhibit oxidoreductase activity acting on CH-NH group donors with NAD or NADP as acceptor, or are directly involved in generating the required cofactors.
| Gene | Major Role | Research Relevance |
|---|---|---|
| NADK | NAD kinase; phosphorylates NAD+ to NADP+ | Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis |
| NADK2 | Mitochondrial NAD kinase; generates NADP(H) | Essential for proline biosynthesis and mitochondrial fatty acid synthesis |
| NNMT | Nicotinamide N-methyltransferase; consumes SAM and produces N-methylnicotinamide | Knockdown protects against diet-induced obesity |
| MTHFD2 | Methylenetetrahydrofolate dehydrogenase; uses NAD+ or NADP+ | Involved in one-carbon metabolism and nucleotide synthesis |
| ALDH | Aldehyde dehydrogenase; oxidizes aldehydes using NAD(P)+ | Redox balance and detoxification |
| GLUD1 | Glutamate dehydrogenase; converts glutamate to α-ketoglutarate using NAD(P)+ | Amino acid metabolism and ammonia detoxification |
| PRODH | Proline dehydrogenase; oxidizes proline using NAD+ | Proline catabolism and redox signaling |
| PYCR1 | Pyrroline-5-carboxylate reductase; reduces P5C to proline using NAD(P)H | Proline biosynthesis; mitochondrial NADPH-dependent |
| FASN | Fatty acid synthase; uses NADPH for fatty acid synthesis | Lipogenesis; linked to NADPH supply |
| IDH1 | Isocitrate dehydrogenase 1 (cytosolic); produces NADPH | Cancer metabolism and redox regulation |
| IDH2 | Isocitrate dehydrogenase 2 (mitochondrial); produces NADPH | Mitochondrial redox balance and lipoylation |
| G6PD | Glucose-6-phosphate dehydrogenase; produces NADPH | Pentose phosphate pathway and antioxidant defense |
| ME1 | Malic enzyme 1; produces NADPH | Lipogenesis and redox homeostasis |
| SHMT2 | Serine hydroxymethyltransferase 2; uses NADP+ | One-carbon metabolism and mitochondrial folate cycle |
| MTHFD1L | Methylenetetrahydrofolate dehydrogenase 1-like; uses NADP+ | Mitochondrial folate metabolism |
| NADPH oxidase | Produces reactive oxygen species using NADPH | Immune defense and oxidative stress |
| CCR4C | Chloroplast-localized NADP(H) phosphatase | Regulates NAD(P)(H) balance in Arabidopsis |
How Is oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor Regulated?
The activity of enzymes in GO:0016646 is regulated at multiple levels. Transcriptional regulation controls the expression of genes like NADK, NADK2, and NNMT in response to metabolic demands. Post-translational modifications, such as phosphorylation, can modulate enzyme activity; for example, human NAD kinase is regulated by its N-terminal domain and possibly by phosphorylation. Allosteric regulation by NAD(P)+/NADPH ratios provides feedback control to maintain redox homeostasis. In mitochondria, NADK2 activity is essential for supplying NADP(H) for proline biosynthesis and fatty acid synthesis, and its loss impairs these pathways. Additionally, the availability of NAD+ and NADP+ precursors (e.g., nicotinamide, tryptophan) influences flux through these reactions.
oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| NNMT | Obesity and metabolic syndrome | Knockout mouse or knockdown in adipocytes |
| NADK | Cancer and folate metabolism | Conditional knockout in cancer cell lines |
| NADK2 | Mitochondrial dysfunction and proline biosynthesis defects | NADK2 knockout cells and mouse models |
| MTHFD2 | Cancer and one-carbon metabolism | CRISPR knockout in tumor cells |
| IDH1/2 | Glioma and leukemia (oncometabolite production) | Point mutation knock-in models |
Metabolic Disorders and Obesity
Dysregulation of NAD(P)-dependent oxidoreductases is linked to metabolic disorders. Nicotinamide N-methyltransferase (NNMT) knockdown protects against diet-induced obesity in mice, highlighting the role of NAD+ salvage and methylation in energy balance. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis, and its loss impairs proliferation under metabolic stress, suggesting implications for metabolic diseases.
Cancer Metabolism
Cancer cells often reprogram NAD(P) metabolism to support rapid proliferation. NADPH is required for fatty acid synthesis and antioxidant defense, and enzymes like NADK and MTHFD2 are upregulated in various cancers. Targeting these pathways is a potential therapeutic strategy. Mitochondrial NADP(H) generation by NADK2 supports proline biosynthesis, which can be critical for cancer cell survival under stress.
Mitochondrial Dysfunction
Mitochondrial NADPH fuels fatty acid synthesis and lipoylation, which are essential for oxidative metabolism. Defects in NADK2 or related enzymes can lead to mitochondrial dysfunction and metabolic disorders. The redox balance maintained by these reactions is crucial for mitochondrial health.
From oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of NADK affect folate-dependent nucleotide synthesis? | NADK knockout cell lines |
| Does NNMT knockdown protect against diet-induced obesity? | NNMT knockout mouse |
| Is mitochondrial NADPH essential for proline biosynthesis? | NADK2 knockout cells |
| How does NADK2 support fatty acid synthesis and lipoylation? | NADK2 knockout mitochondria |
| What is the role of CCR4C in chloroplast NADP(H) balance? | CCR4C knockout Arabidopsis |
| Can point mutations in NADK alter its activity? | Knock-in of specific NADK mutations |
How to Study the oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NAD(P)H absorbance assay | Enzyme activity by NAD(P)H production | Kinetic studies of dehydrogenases |
| LC-MS metabolomics | NAD(P)+/NAD(P)H levels and pathway intermediates | Redox balance and metabolic flux |
| CRISPR knockout | Loss-of-function phenotypes | Gene function in metabolism |
| Cryo-EM | Protein structure at near-atomic resolution | Mechanistic studies of NADK |
| Isotope tracing | Flux through metabolic pathways | Proline and folate synthesis |
| Western blot | Protein expression levels | Validation of knockout/knockdown |
| Immunofluorescence | Subcellular localization | Mitochondrial vs cytosolic NADK |
Enzymatic Activity Assays
Direct measurement of oxidoreductase activity using purified enzymes or cell lysates. These assays monitor the reduction of NAD+ or NADP+ to NADH or NADPH by measuring absorbance at 340 nm. They are used to confirm the function of candidate genes and to test inhibitors.
Metabolomics and Flux Analysis
Mass spectrometry-based metabolomics can quantify NAD(P)+/NAD(P)H ratios and intermediates of pathways like proline and folate metabolism. Isotope tracing can reveal flux through these reactions.
Genetic Knockout and Knockdown
CRISPR-Cas9 knockout or RNAi knockdown of genes like NADK, NADK2, and NNMT in cell lines or animal models to study their roles in metabolism and disease.
Structural Biology
Cryo-EM and X-ray crystallography to determine the structure of enzymes like human NAD kinase, revealing regulatory domains and catalytic mechanisms.
How CRISPR Can Be Used to Study GO:0016646 oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor
Knockout
CRISPR-Cas9 knockout of genes encoding enzymes with GO:0016646 activity (e.g., NADK, NADK2, NNMT) is used to study loss-of-function phenotypes. For example, NADK knockout impairs folate-dependent nucleotide synthesis, and NNMT knockout protects against diet-induced obesity.
Point Mutation
Point mutations can be introduced to alter catalytic residues or regulatory sites. For instance, mutations in NADK's N-terminal domain affect its regulation. Such models help dissect enzyme mechanism and regulation.
Knock-in
Knock-in of tagged versions (e.g., FLAG, GFP) allows for affinity purification and localization studies. Knock-in of disease-associated mutations can model human disorders.
Overexpression
Overexpression of genes like NADK or NNMT can reveal gain-of-function effects on metabolism and disease. For example, NNMT overexpression promotes obesity.
How EDITGENE Supports oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor Research
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Frequently Asked Questions About oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor
What is GO:0016646?
GO:0016646 is a Gene Ontology molecular function term for oxidoreductase activity where a CH-NH group donates electrons to NAD+ or NADP+, reducing them to NADH or NADPH.
What genes are involved in oxidoreductase activity, acting on the CH-NH group of donors, NAD or NADP as acceptor?
Key genes include NADK, NADK2, NNMT, MTHFD2, GLUD1, and PYCR1, among others.
What is the role of NADK in this activity?
NADK phosphorylates NAD+ to NADP+, providing the cofactor for NADP-dependent oxidoreductases.
How is mitochondrial NADPH generated?
Mitochondrial NADPH is generated by NADK2, which phosphorylates NAD+ to NADP+, and by enzymes like IDH2 and MTHFD2.
What diseases are linked to this activity?
Dysregulation is linked to obesity, cancer, and mitochondrial dysfunction.
Can CRISPR be used to study this activity?
Yes, CRISPR knockout, point mutation, and knock-in models are powerful tools to study gene function in this pathway.
What is the connection to proline biosynthesis?
Mitochondrial NADP(H) generated by NADK2 is essential for proline biosynthesis, as PYCR1 uses NADPH to reduce P5C to proline.
How does NNMT relate to obesity?
NNMT knockdown protects against diet-induced obesity, likely by altering NAD+ salvage and methylation potential.
What methods are used to measure this activity?
Enzymatic assays measuring NAD(P)H absorbance, metabolomics, and isotope tracing are commonly used.
Is this activity important in plants?
Yes, NADP(H) balance regulated by CCR4C is critical for chloroplast function and photosynthesis.
Conclusion
GO:0016646 represents a fundamental class of redox reactions that sustain cellular metabolism by generating NADH and NADPH. These cofactors are indispensable for biosynthesis, antioxidant defense, and energy production. Research into the enzymes and pathways associated with this activity has revealed critical roles in proline biosynthesis, folate metabolism, and disease states such as obesity and cancer. Understanding the regulation and function of these enzymes offers opportunities for therapeutic intervention and metabolic engineering.
References
- 1. Zhu J et al.. 2021. Mitochondrial NADP(H) generation is essential for proline biosynthesis.. Science 372(6545):968-972 PMID: 33888598
- 2. Kraus D et al.. 2014. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.. Nature 508(7495):258-62 PMID: 24717514
- 3. Kim D et al.. 2025. Mitochondrial NADPH fuels mitochondrial fatty acid synthesis and lipoylation to power oxidative metabolism.. Nat Cell Biol 27(5):790-800 PMID: 40258949
- 4. McGuinness ET et al.. 1985. NAD+ kinase--a review.. Int J Biochem 17(1):1-11 PMID: 2987053
- 5. Praharaj PP et al.. 2025. Cryo-EM structure and regulation of human NAD kinase.. Sci Adv 11(4):eads2664 PMID: 39854463
- 6. Arnon DI. 1971. The light reactions of photosynthesis.. Proc Natl Acad Sci U S A 68(11):2883-92 PMID: 4400251
- 7. Flickinger KM et al.. 2025. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.. Nat Metab 7(6):1150-1167 PMID: 40316835
- 8. Akashi K et al.. 2025. Identification of CCR4C as a chloroplast-localized NADP(H) phosphatase regulating NAD(P)(H) balance in Arabidopsis.. Proc Natl Acad Sci U S A 122(42):e2504605122 PMID: 41091769
