GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004487 describes the NAD+-dependent oxidation of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate to (6R)-5,10-methenyltetrahydrofolate, a key one-carbon folate interconversion [1,4].
• The enzyme is typically a bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (MTHFD2 in humans) that is mitochondrially targeted in transformed cells [4,8].
• NAD-dependent activity is expressed by immortal and oncogene-transformed cells, making it a cancer-associated metabolic node [1,3].
• In oral squamous cell carcinoma, NAD-dependent MTHFD2 inhibits proliferation and promotes apoptosis, indicating context-dependent roles.
• Magnesium and phosphate ions enable NAD binding to the bifunctional enzyme, linking metal and anion homeostasis to its catalysis.
• The NAD- and NADP-dependent mitochondrial enzymes can rescue mthfd2 null fibroblasts, revealing functional redundancy in one-carbon metabolism.
Description
GO:0004487, methylenetetrahydrofolate dehydrogenase (NAD+) activity, is a molecular function that catalyzes the NAD+-dependent conversion of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate to (6R)-5,10-methenyltetrahydrofolate [1,4]. This reaction is a central node in one-carbon metabolism, supplying methylene and formyl units for nucleotide biosynthesis and amino acid interconversion [4,6]. The enzyme is often isolated as a bifunctional protein with methenyltetrahydrofolate cyclohydrolase activity, and in transformed cells it is mitochondrially targeted [4,8]. Researchers study this activity because it is differentially expressed in normal versus neoplastic cells and is induced by oncogenic transformation [1,3]. Its kinetic independence from the cyclohydrolase domain and its requirement for magnesium and phosphate ions for NAD binding make it a distinctive target for mechanistic and structural studies [6,7]. In oral squamous cell carcinoma, modulation of the NAD-dependent enzyme affects proliferation and apoptosis, underscoring its potential as a disease-relevant metabolic function.
methylenetetrahydrofolate dehydrogenase (NAD+) activity At A Glance
| GO ID | GO:0004487 |
|---|---|
| GO term | methylenetetrahydrofolate dehydrogenase (NAD+) activity |
| Ontology | molecular_function |
| Synonym | 5,10-methylenetetrahydrofolate dehydrogenase activity; 5,10-methylenetetrahydrofolate:NAD+ oxidoreductase |
| Major function | NAD+-dependent oxidation of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate to (6R)-5,10-methenyltetrahydrofolate |
| Cofactor | NAD+ (nicotinamide adenine dinucleotide, oxidized form) |
| Substrate | (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate |
| Product | (6R)-5,10-methenyltetrahydrofolate and NADH |
| Associated enzyme | Bifunctional methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (e.g., MTHFD2 in humans) |
| Ion requirement | Magnesium and phosphate ions enable NAD binding |
| Subcellular localization | Mitochondrial in transformed cells |
What Is GO:0004487?
GO:0004487 is defined by the QuickGO as the catalysis of the reaction: (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NAD+ = (6R)-5,10-methenyltetrahydrofolate + NADH. In other words, it is the NAD+-dependent oxidation of a methylene-substituted tetrahydrofolate to a methenyl-substituted tetrahydrofolate, producing NADH [1,4]. This activity is synonymous with 5,10-methylenetetrahydrofolate dehydrogenase activity and 5,10-methylenetetrahydrofolate:NAD+ oxidoreductase. It is distinct from the NADP-dependent isozyme and is often found as part of a bifunctional enzyme with cyclohydrolase activity [4,6].
Why Is methylenetetrahydrofolate dehydrogenase (NAD+) activity Important in Cell Biology?
GO:0004487 is important because it links one-carbon folate metabolism to redox balance and nucleotide precursor supply, and its NAD-dependent form is selectively expressed in immortal and oncogene-transformed cells [1,3]. This makes it a potential metabolic marker and therapeutic target in cancer, where its activity can influence proliferation and apoptosis. The bifunctional nature of the enzyme and its mitochondrial localization in transformed cells further connect it to compartmentalized folate metabolism and mitochondrial one-carbon flux [4,8]. Understanding this activity is therefore relevant to cancer metabolism, drug discovery, and the basic biochemistry of folate interconversion [5,6].
• Provides a key step in one-carbon metabolism by converting methylene-tetrahydrofolate to methenyl-tetrahydrofolate [1,4].
• Is expressed by immortal cells and oncogene-transformed cells, suggesting a role in cellular immortalization and transformation [1,3].
• In oral squamous cell carcinoma, the NAD-dependent enzyme inhibits proliferation and promotes apoptosis, indicating tumor-suppressive-like behavior in this context.
• The bifunctional enzyme couples dehydrogenase and cyclohydrolase activities, which are kinetically independent.
• Magnesium and phosphate ions are required for NAD binding, linking ion homeostasis to enzyme function.
• Mitochondrial targeting in transformed cells places the activity in a distinct metabolic compartment.
• NAD- and NADP-dependent mitochondrial enzymes can rescue mthfd2 null fibroblasts, showing redundancy in one-carbon metabolism.
• The activity is a potential target for metabolic inhibitors in cancer research [1,2].
• Its differential expression between normal and neoplastic cells makes it a candidate biomarker.
• Studying it helps clarify how folate metabolism supports nucleotide biosynthesis in proliferating cells [4,6].
What Happens During methylenetetrahydrofolate dehydrogenase (NAD+) activity?
Substrate binding and NAD+ interaction
In simple terms: The enzyme first grabs its folate substrate and the NAD+ cofactor.
The reaction begins with binding of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate and NAD+ to the bifunctional enzyme. Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase, as shown for the bifunctional enzyme. This step is essential for orienting the cofactor for hydride transfer.
Hydride transfer and oxidation
In simple terms: The enzyme removes a hydride from the folate substrate and gives it to NAD+.
The catalytic step involves oxidation of the methylene group of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate to a methenyl group, with concomitant reduction of NAD+ to NADH. This NAD-dependent dehydrogenase activity is the defining reaction of GO:0004487 [1,4]. The reaction is reversible in principle, but the dehydrogenase and cyclohydrolase activities of the bifunctional enzyme are kinetically independent.
Product formation and cyclohydrolase coupling
In simple terms: The product is a methenyl-folate, which can be further processed by the cyclohydrolase part of the same enzyme.
The immediate product is (6R)-5,10-methenyltetrahydrofolate and NADH. In the bifunctional enzyme, the methenyltetrahydrofolate cyclohydrolase domain can subsequently hydrolyze the methenyl group to formyl-tetrahydrofolate, linking the dehydrogenase step to downstream one-carbon pools [4,6]. This coupling is typical of the mitochondrial enzyme in transformed cells.
Mitochondrial context and cellular role
In simple terms: In transformed cells, this activity happens inside mitochondria and supports one-carbon metabolism there.
NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme. This localization places the activity in a compartment that supplies one-carbon units for mitochondrial processes and can influence overall cellular folate metabolism. The NAD- and NADP-dependent mitochondrial enzymes can rescue mthfd2 null fibroblasts, indicating overlapping functions in mitochondrial one-carbon metabolism.
Key Genes Involved in GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity
The following genes and proteins are directly or functionally associated with GO:0004487, based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTHFD2 | Mitochondrial bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase with NAD-dependent activity | Expressed in transformed cells; target for cancer metabolism studies [4,8] |
| MTHFD2L | Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase | Can rescue mthfd2 null fibroblasts, showing redundancy |
| MTHFD1 | Cytoplasmic trifunctional enzyme with NADP-dependent dehydrogenase activity | Related folate interconversion, but distinct cofactor specificity |
| MTHFD1L | Mitochondrial monofunctional formyltetrahydrofolate synthetase | Downstream one-carbon metabolism, not directly GO:0004487 |
| MTHFR | Methylenetetrahydrofolate reductase | Competes for methylene-tetrahydrofolate, influencing folate flux |
| SHMT1 | Serine hydroxymethyltransferase, cytoplasmic | Generates methylene-tetrahydrofolate, substrate for GO:0004487 |
| SHMT2 | Serine hydroxymethyltransferase, mitochondrial | Generates methylene-tetrahydrofolate in mitochondria |
| TYMS | Thymidylate synthase | Consumes methylene-tetrahydrofolate, linked to one-carbon pools |
| DHFR | Dihydrofolate reductase | Regenerates tetrahydrofolate, supporting folate cycle |
| GART | Phosphoribosylglycinamide formyltransferase | Uses formyl-tetrahydrofolate from one-carbon metabolism |
| ATIC | AICAR transformylase | Uses formyl-tetrahydrofolate in purine synthesis |
| MTR | Methionine synthase | Links folate cycle to methionine cycle |
| MAT2A | Methionine adenosyltransferase | Produces SAM, connected to methylation |
| GLS | Glutaminase | Supports mitochondrial one-carbon metabolism |
| IDH2 | Isocitrate dehydrogenase 2 | Mitochondrial NADPH source, related to redox |
| SLC25A32 | Mitochondrial folate transporter | Imports folate into mitochondria for one-carbon metabolism |
How Is methylenetetrahydrofolate dehydrogenase (NAD+) activity Regulated?
The activity of NAD-dependent methylenetetrahydrofolate dehydrogenase is regulated at multiple levels. Its expression is induced in immortal and oncogene-transformed cells, suggesting transcriptional or post-transcriptional upregulation during transformation [1,3]. The bifunctional enzyme requires magnesium and phosphate ions for NAD binding, so ion availability can modulate its activity. The dehydrogenase and cyclohydrolase activities are kinetically independent, meaning that substrate flux through one activity does not necessarily dictate the other. Mitochondrial localization in transformed cells may also subject the enzyme to compartment-specific regulation by NAD+ availability and one-carbon demand. Additionally, redundancy with NADP-dependent mitochondrial enzymes can compensate for loss of function, as shown by rescue of mthfd2 null fibroblasts.
methylenetetrahydrofolate dehydrogenase (NAD+) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTHFD2 | Cancer metabolism, oral squamous cell carcinoma proliferation and apoptosis | MTHFD2 knockout or overexpression in OSCC cell lines |
| MTHFD2 | Oncogene-transformed cell growth | Oncogene-transformed fibroblasts with MTHFD2 knockdown [1,3] |
| MTHFD2L | Mitochondrial one-carbon metabolism redundancy | Mthfd2 null fibroblasts rescued with MTHFD2L |
| MTHFD2 | Mitochondrial folate metabolism in transformed cells | Mitochondrial isolation from transformed cells |
| MTHFD2 | NAD binding and ion dependence | Enzyme kinetics with magnesium and phosphate variation |
Cancer metabolism and transformation
NAD-dependent methylenetetrahydrofolate dehydrogenase activity is expressed by immortal cells and oncogene-transformed cells, and its levels differ between normal tissue and neoplastic cells [1,3]. This suggests a role in supporting the metabolic demands of rapidly proliferating cells. In oral squamous cell carcinoma, the NAD-dependent enzyme inhibits cell proliferation and promotes apoptosis, indicating that its role may be context-dependent and potentially tumor-suppressive in this cancer type.
Mitochondrial one-carbon metabolism in disease
The mitochondrial localization of the enzyme in transformed cells links GO:0004487 to mitochondrial one-carbon metabolism, which is critical for nucleotide biosynthesis and redox homeostasis. Disruption of this pathway can affect cell survival, and redundancy with NADP-dependent enzymes may influence disease phenotypes. Understanding these mitochondrial roles could inform therapies targeting cancer cell metabolism.
Folate-related pathologies
Because the enzyme interconverts folate species, its activity is part of the broader folate cycle that, when perturbed, is associated with developmental and hematological disorders. However, direct disease associations for GO:0004487 specifically are not well established in the verified literature, and most evidence comes from cancer cell models [1,2,4].
From methylenetetrahydrofolate dehydrogenase (NAD+) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of NAD-dependent dehydrogenase activity affect proliferation? | MTHFD2 knockout cell lines (e.g., cancer cell lines) |
| Can NADP-dependent enzymes compensate for loss of GO:0004487? | Mthfd2 null fibroblasts rescued with MTHFD2L or MTHFD1 |
| How does mitochondrial localization influence function? | Tagged knock-in of MTHFD2 with mitochondrial targeting signal |
| What is the kinetic independence of dehydrogenase and cyclohydrolase? | Point mutations in bifunctional enzyme domains |
| How do ions regulate NAD binding? | Enzyme assays with varying Mg2+ and phosphate |
| Is the activity induced by oncogenic transformation? | Oncogene-transformed cell models compared to normal cells [1,3] |
How to Study the methylenetetrahydrofolate dehydrogenase (NAD+) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NADH absorbance assay | Dehydrogenase activity via NADH production | Enzyme kinetics and inhibitor testing [4,6] |
| Subcellular fractionation | Mitochondrial vs cytoplasmic localization | Confirming mitochondrial targeting |
| RNA-seq / qPCR | Expression levels of MTHFD2 and related genes | Comparing normal vs transformed cells [1,3] |
| 13C metabolic tracing | Flux through one-carbon metabolism | Assessing pathway activity and redundancy |
| Western blot | Protein levels and post-translational modifications | Validating knockout or overexpression |
| Immunofluorescence | Subcellular localization in situ | Visualizing mitochondrial co-localization |
| Site-directed mutagenesis | Kinetic independence of domains | Dissecting dehydrogenase vs cyclohydrolase |
| Ion titration assays | Effect of Mg2+ and phosphate on NAD binding | Characterizing cofactor requirements |
Enzymatic activity assays
Direct measurement of GO:0004487 can be performed by monitoring NADH production at 340 nm using purified enzyme or cell lysates. Such assays have been used to characterize the bifunctional enzyme from ascites tumor cells and to demonstrate kinetic independence of the dehydrogenase and cyclohydrolase activities [4,6]. Ion dependence can be tested by varying magnesium and phosphate concentrations.
Subcellular fractionation and imaging
To determine mitochondrial localization, subcellular fractionation followed by immunoblotting or fluorescence microscopy with mitochondrial markers can be used. This approach established that the NAD-dependent enzyme in transformed cells is mitochondrial. Tagged knock-in models can further confirm localization in live cells.
Expression profiling in cancer models
RNA-seq and quantitative PCR can compare expression of MTHFD2 and related genes between normal and neoplastic cells or upon oncogene transformation [1,3]. In oral squamous cell carcinoma, functional studies used knockdown and overexpression to link the enzyme to proliferation and apoptosis.
Metabolic flux analysis
Stable isotope tracing with 13C-labeled serine or formate can quantify one-carbon flux through the folate cycle. Such methods help assess how loss or gain of GO:0004487 activity affects downstream nucleotide synthesis and redox balance, as suggested by rescue experiments in mthfd2 null fibroblasts.
How CRISPR Can Be Used to Study GO:0004487 methylenetetrahydrofolate dehydrogenase (NAD+) activity
Knockout
CRISPR knockout of MTHFD2 can eliminate GO:0004487 activity in cells, allowing researchers to test its role in proliferation, apoptosis, and one-carbon flux. Such models are valuable for validating findings from knockdown studies in cancer cells. Knockout of MTHFD2 in fibroblasts can be rescued by NADP-dependent enzymes, highlighting redundancy.
Point Mutation
Point mutations can be introduced into the catalytic domain of MTHFD2 to dissect the dehydrogenase activity from the cyclohydrolase activity, which are kinetically independent. Mutations affecting NAD binding or ion coordination can clarify the roles of magnesium and phosphate.
Knock-in
Knock-in of tagged MTHFD2 (e.g., FLAG or GFP) enables localization and interaction studies. This is particularly useful for confirming mitochondrial targeting in transformed cells. Knock-in of disease-associated variants can model altered enzyme function.
Overexpression
Overexpression of MTHFD2 can increase NAD-dependent dehydrogenase activity and has been used to study its effects on proliferation and apoptosis in oral squamous cell carcinoma. Overexpression in normal cells can test whether the activity is sufficient to induce transformation-associated phenotypes [1,3].
How EDITGENE Supports methylenetetrahydrofolate dehydrogenase (NAD+) activity Research
Researchers studying methylenetetrahydrofolate dehydrogenase (NAD+) activity-related genes often need to determine whether a candidate gene is causally involved in metabolic reprogramming, proliferation, or apoptosis. EDITGENE provides CRISPR-based cell model services to enable such functional studies with high specificity and reproducibility.
Contact EDITGENE today to design your custom CRISPR model for methylenetetrahydrofolate dehydrogenase (NAD+) activity research.
Frequently Asked Questions About methylenetetrahydrofolate dehydrogenase (NAD+) activity
What is GO:0004487?
GO:0004487 is the Gene Ontology molecular function term for methylenetetrahydrofolate dehydrogenase (NAD+) activity, which catalyzes the NAD+-dependent conversion of (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate to (6R)-5,10-methenyltetrahydrofolate [1,4].
What genes are involved in methylenetetrahydrofolate dehydrogenase (NAD+) activity?
The primary gene is MTHFD2, which encodes a mitochondrial bifunctional enzyme with this activity [4,8]. MTHFD2L is a related gene that can also provide NAD-dependent activity.
What is the reaction catalyzed by GO:0004487?
The reaction is: (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NAD+ = (6R)-5,10-methenyltetrahydrofolate + NADH [1,4].
Is methylenetetrahydrofolate dehydrogenase (NAD+) activity mitochondrial?
In transformed cells, the NAD-dependent enzyme is mitochondrially targeted, as shown by subcellular fractionation.
How is this activity related to cancer?
It is expressed by immortal and oncogene-transformed cells, and in oral squamous cell carcinoma it inhibits proliferation and promotes apoptosis [1,2,3].
What cofactors are required for GO:0004487?
NAD+ is the direct electron acceptor, and magnesium and phosphate ions enable NAD binding to the bifunctional enzyme.
Can NADP-dependent enzymes replace NAD-dependent methylenetetrahydrofolate dehydrogenase?
Yes, NAD- and NADP-dependent mitochondrially targeted enzymes can rescue mthfd2 null fibroblasts, indicating functional redundancy.
What methods are used to study methylenetetrahydrofolate dehydrogenase (NAD+) activity?
Common methods include NADH absorbance assays, subcellular fractionation, RNA-seq, and metabolic flux analysis [4,6,8].
Are the dehydrogenase and cyclohydrolase activities coupled?
They are kinetically independent in the bifunctional enzyme from ascites tumor cells.
What CRISPR models are available for studying GO:0004487?
Knockout, point mutation, knock-in, and overexpression models of MTHFD2 and related genes can be generated to study this activity [2,5,6].
Conclusion
GO:0004487, methylenetetrahydrofolate dehydrogenase (NAD+) activity, is a central enzymatic step in one-carbon folate metabolism with particular relevance to transformed and immortal cells. Its bifunctional nature, mitochondrial localization, and ion-dependent NAD binding make it a distinctive target for mechanistic and cancer metabolism research [4,6,7,8]. Functional studies in oral squamous cell carcinoma and other models highlight its potential to influence proliferation and apoptosis, though its roles are context-dependent [1,2,3]. Continued investigation using CRISPR-based models and metabolic profiling will clarify how this activity contributes to health and disease.
References
- 1. Smith GK et al.. 1990. Activity of an NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase in normal tissue, neoplastic cells, and oncogene-transformed cells.. Arch Biochem Biophys 283(2):367-71 PMID: 2275549
- 2. Zhao L et al.. 2021. NAD-dependent methylenetetrahydrofolate dehydrogenase inhibits oral squamous cell carcinoma cell proliferation and promotes apoptosis.. Transl Cancer Res 10(3):1457-1469 PMID: 35116471
- 3. Mejia NR et al.. 1985. NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells.. J Biol Chem 260(27):14616-20 PMID: 3877056
- 4. Mejia NR et al.. 1986. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells. Purification and properties.. J Biol Chem 261(20):9509-13 PMID: 3487546
- 5. Patel H et al.. 2005. NAD- and NADP-dependent mitochondrially targeted methylenetetrahydrofolate dehydrogenase-cyclohydrolases can rescue mthfd2 null fibroblasts.. Arch Biochem Biophys 442(1):133-9 PMID: 16150419
- 6. Rios-Orlandi EM et al.. 1988. The activities of the NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells are kinetically independent.. J Biol Chem 263(10):4662-7 PMID: 3258307
- 7. Christensen KE et al.. 2005. Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.. J Biol Chem 280(40):34316-23 PMID: 16100107
- 8. Mejia NR et al.. 1988. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme.. Biochem Biophys Res Commun 155(1):1-6 PMID: 3261979
