GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106313 describes the NADPH-dependent catalytic activity of methylenetetrahydrofolate reductase (MTHFR), which converts (6S)-5-methyl-5,6,7,8-tetrahydrofolate to (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate while oxidizing NADPH to NADP+.
• MTHFR is a central enzyme in one-carbon metabolism, linking folate and methionine cycles and influencing homocysteine levels.
• Common MTHFR polymorphisms, such as C677T, reduce enzyme activity and are associated with diverse clinical phenotypes including vascular, neurological, and pregnancy-related conditions.
• MTHFR activity is relevant beyond humans; in Mycobacterium tuberculosis, decreased MTHFR activity increases sensitivity to para-aminosalicylic acid.
• Experimental models for studying MTHFR include knockout, point-mutation, knock-in, and overexpression cell lines, as well as biochemical assays.
• Understanding GO:0106313 helps researchers design CRISPR-based models to dissect MTHFR function in health and disease.
Description
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, and its NADPH-dependent activity is captured by the Gene Ontology term GO:0106313. This molecular function catalyzes the conversion of (6S)-5-methyl-5,6,7,8-tetrahydrofolate to (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate, using NADP+ as an electron acceptor and producing NADPH and H+. The reaction is essential for maintaining the balance between folate species and for providing methyl groups for methionine synthesis and DNA methylation. Researchers study this activity to understand one-carbon metabolism and its impact on human health. MTHFR polymorphisms, particularly C677T, are among the most studied genetic variants and have been linked to altered enzyme activity and disease risk. In addition to human genetics, MTHFR activity is a target in infectious diseases; for example, in Mycobacterium tuberculosis, reduced MTHFR activity leads to increased sensitivity to para-aminosalicylic acid. Thus, GO:0106313 represents a critical biochemical function with broad implications for genetics, metabolism, and pharmacology.
methylenetetrahydrofolate reductase (NADPH) activity At A Glance
| GO ID | GO:0106313 |
|---|---|
| GO term | methylenetetrahydrofolate reductase (NADPH) activity |
| Ontology | molecular_function |
| Synonym | (none) |
| Major function | Catalyzes the NADP+-dependent conversion of (6S)-5-methyl-5,6,7,8-tetrahydrofolate to (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate |
| Reaction direction | Reversible; physiological direction depends on cellular conditions |
| Cofactor | NADP+/NADPH |
| Substrates | (6S)-5-methyl-5,6,7,8-tetrahydrofolate and NADP+ |
| Products | (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate, NADPH, and H+ |
| Associated gene | MTHFR (human) |
What Is GO:0106313?
GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity is defined as the catalysis of the reaction: (6S)-5-methyl-5,6,7,8-tetrahydrofolate + NADP+ = (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + NADPH + H+. In simpler terms, it is the enzyme activity that interconverts two forms of folate using NADP+ as a cofactor, thereby regulating the availability of methyl groups for various cellular processes.
Why Is methylenetetrahydrofolate reductase (NADPH) activity Important in Cell Biology?
GO:0106313 is important because MTHFR activity sits at the intersection of folate and methionine metabolism, influencing homocysteine remethylation, nucleotide synthesis, and DNA methylation. Altered MTHFR activity, often due to genetic polymorphisms, has been associated with a wide range of conditions including cardiovascular disease, neural tube defects, psychiatric disorders, and pregnancy complications such as preeclampsia. Moreover, MTHFR is a potential drug target in pathogens; in Mycobacterium tuberculosis, decreased MTHFR activity increases susceptibility to para-aminosalicylic acid. Therefore, studying this activity provides insights into basic metabolism and translational opportunities.
• MTHFR activity regulates the balance between folate species, affecting methionine synthesis and DNA methylation.
• The C677T polymorphism in MTHFR reduces enzyme activity and is linked to hyperhomocysteinemia and vascular disease.
• MTHFR activity is associated with psychiatric disorders, including depression and schizophrenia.
• In women with preeclampsia, decreased MTHFR activity and elevated homocysteine have been observed.
• MTHFR activity influences thrombophilia risk, contributing to hypercoagulable states.
• In Mycobacterium tuberculosis, MTHFR activity modulates sensitivity to para-aminosalicylic acid.
• MTHFR missense variants can have diverse effects on enzyme function, as revealed by deep mutational scanning.
• Understanding MTHFR activity aids in interpreting genetic testing and personalized medicine.
• MTHFR is a target for nutritional and pharmacological interventions.
• CRISPR-based models enable precise dissection of MTHFR function in disease.
Molecular Mechanism of methylenetetrahydrofolate reductase (NADPH) activity
Substrate Binding and Catalysis
In simple terms: The enzyme grabs a methylated folate molecule and NADP+ to start the reaction.
MTHFR binds (6S)-5-methyl-5,6,7,8-tetrahydrofolate and NADP+ in its active site. The enzyme catalyzes the transfer of a methylene group, converting the substrate to (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate while reducing NADP+ to NADPH and releasing H+. This reaction is reversible, but in vivo it typically proceeds in the direction of 5-methyl-THF formation for methionine synthesis.
Role of NADPH/NADP+ Cofactor
In simple terms: NADPH acts like a rechargeable battery that helps the enzyme work.
NADPH is a critical cofactor for MTHFR activity. The enzyme uses NADPH as an electron donor in the reverse reaction, but the GO term specifically describes the NADP+-dependent oxidation of 5-methyl-THF. The ratio of NADPH to NADP+ can influence enzyme activity and directionality.
Allosteric Regulation by SAM
In simple terms: A molecule called SAM can tell the enzyme to slow down or speed up.
MTHFR is allosterically inhibited by S-adenosylmethionine (SAM), the primary methyl donor. When SAM levels are high, MTHFR activity is reduced, preventing excessive methyl group production. Conversely, low SAM levels activate the enzyme. This feedback mechanism ensures balanced one-carbon metabolism.
Genetic Variants Affecting Activity
In simple terms: Small changes in the gene can make the enzyme work better or worse.
Common polymorphisms such as C677T (Ala222Val) and A1298C (Glu429Ala) reduce MTHFR activity. The C677T variant is associated with decreased enzyme stability and increased thermolability, leading to elevated homocysteine levels. Deep mutational scanning has revealed that different missense variants have varying effects on MTHFR function, highlighting the importance of genotype-phenotype correlations.
Tissue-Specific Expression and Isoforms
In simple terms: The enzyme can be made in different forms in different parts of the body.
MTHFR is expressed in most tissues, with highest levels in liver, kidney, and brain. Alternative splicing produces multiple transcript variants, though their functional significance is not fully understood. Tissue-specific differences in MTHFR activity may contribute to variable disease manifestations.
Key Genes Involved in GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity
The following genes and proteins are directly or indirectly involved in methylenetetrahydrofolate reductase (NADPH) activity and its metabolic context.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTHFR | Encodes the enzyme with GO:0106313 activity | Central to folate metabolism; polymorphisms linked to disease |
| MTR | Methionine synthase; uses 5-methyl-THF product | Connects MTHFR activity to methionine synthesis |
| MTRR | Methionine synthase reductase; regenerates MTR | Supports methionine cycle |
| CBS | Cystathionine beta-synthase; transsulfuration | Links homocysteine to cysteine synthesis |
| MTHFD1 | Methylenetetrahydrofolate dehydrogenase | Provides 5,10-methylene-THF for MTHFR reaction |
| SHMT1 | Serine hydroxymethyltransferase | Generates 5,10-methylene-THF |
| SHMT2 | Mitochondrial serine hydroxymethyltransferase | Contributes to one-carbon metabolism |
| TYMS | Thymidylate synthase; uses 5,10-methylene-THF | Competes with MTHFR for substrate |
| DHFR | Dihydrofolate reductase | Regenerates THF for folate cycle |
| MTHFD2 | Mitochondrial methylenetetrahydrofolate dehydrogenase | Supports mitochondrial one-carbon flux |
| GART | Phosphoribosylglycinamide formyltransferase | Purine synthesis; uses folate derivatives |
| ATIC | AICAR transformylase | Purine synthesis; folate-dependent |
| SLC19A1 | Reduced folate carrier | Transports folates into cells |
| FOLR1 | Folate receptor alpha | Mediates folate uptake |
| MAT1A | Methionine adenosyltransferase | Produces SAM, allosteric inhibitor of MTHFR |
| GNMT | Glycine N-methyltransferase | Regulates SAM/SAH ratio |
| AHCY | S-adenosylhomocysteine hydrolase | Controls homocysteine levels |
| BHMT | Betaine-homocysteine methyltransferase | Alternative homocysteine remethylation pathway |
How Is methylenetetrahydrofolate reductase (NADPH) activity Regulated?
MTHFR activity is regulated at multiple levels. Allosterically, S-adenosylmethionine (SAM) inhibits MTHFR, while S-adenosylhomocysteine (SAH) may activate it, providing feedback based on methyl donor status. Phosphorylation by casein kinase II has been reported to modulate MTHFR activity, though the precise mechanisms remain under investigation. Additionally, genetic variants such as C677T affect enzyme stability and activity, and expression levels can vary by tissue and developmental stage.
methylenetetrahydrofolate reductase (NADPH) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTHFR | Hyperhomocysteinemia, thrombosis | Knockout or point-mutation cell lines |
| MTHFR | Preeclampsia | Overexpression or knockdown in trophoblast cells |
| MTHFR | Psychiatric disorders | Neuronal cell lines with C677T knock-in |
| MTHFR | Mycobacterium tuberculosis drug sensitivity | Bacterial MTHFR knockout |
| MTHFR | Neural tube defects | Mouse models or iPSC-derived neural cells |
MTHFR Polymorphisms and Vascular Disease
The C677T polymorphism in MTHFR leads to reduced enzyme activity and elevated homocysteine, a risk factor for cardiovascular disease, thrombosis, and stroke. Meta-analyses have associated the TT genotype with increased risk of venous thromboembolism and coronary artery disease, particularly in individuals with low folate status.
MTHFR in Pregnancy Complications
Decreased MTHFR activity has been observed in women with preeclampsia, a hypertensive disorder of pregnancy. A study found lower serum MTHFR activity and higher homocysteine and malondialdehyde levels in preeclamptic women compared to controls, suggesting a role for oxidative stress and impaired folate metabolism.
MTHFR and Psychiatric Disorders
MTHFR activity influences neurotransmitter synthesis and DNA methylation, processes implicated in psychiatric disorders. The C677T variant has been associated with depression, schizophrenia, and bipolar disorder, though findings are inconsistent across populations. Reduced MTHFR activity may contribute to altered one-carbon metabolism in the brain.
MTHFR as a Drug Target in Tuberculosis
In Mycobacterium tuberculosis, decreased MTHFR activity increases sensitivity to para-aminosalicylic acid (PAS), an anti-tuberculosis drug. This suggests that MTHFR inhibitors could potentiate PAS efficacy, highlighting a non-human application of GO:0106313.
From methylenetetrahydrofolate reductase (NADPH) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does MTHFR loss affect homocysteine levels? | MTHFR knockout cell line (e.g., HepG2) |
| How does C677T variant alter enzyme activity? | Point-mutation knock-in of C677T in HEK293 |
| Can MTHFR overexpression rescue folate deficiency? | Overexpression cell line |
| What is the effect of MTHFR on drug sensitivity? | MTHFR knockout in M. tuberculosis |
| Does MTHFR activity influence DNA methylation? | Knockout and rescue with wild-type or mutant MTHFR |
| Can we screen for modifiers of MTHFR activity? | CRISPR library screening in haploid cells |
How to Study the methylenetetrahydrofolate reductase (NADPH) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | MTHFR catalytic activity | Variant functional characterization |
| PCR-RFLP | C677T genotype | Population studies |
| LC-MS/MS | Folate and homocysteine levels | Metabolic profiling |
| Deep mutational scanning | Effect of missense variants | Variant interpretation |
| CRISPR knockout | Gene function loss | Pathway analysis |
| CRISPR knock-in | Specific mutation effects | Disease modeling |
| Western blot | MTHFR protein levels | Expression analysis |
| Immunoassay | Homocysteine concentration | Clinical diagnostics |
Enzymatic Activity Assays
MTHFR activity can be measured in cell lysates or purified protein using spectrophotometric assays that monitor NADPH oxidation or the formation of 5,10-methylene-THF. These assays are essential for validating the functional impact of genetic variants.
Genotyping and Sequencing
Common polymorphisms like C677T are detected by PCR-RFLP, TaqMan assays, or sequencing. Deep mutational scanning can comprehensively map the effects of missense variants on MTHFR activity.
Metabolite Profiling
Levels of homocysteine, methionine, SAM, SAH, and folate species can be quantified by HPLC, LC-MS/MS, or immunoassays to assess the metabolic consequences of altered MTHFR activity.
CRISPR-Based Functional Genomics
CRISPR knockout, knock-in, and overexpression models allow precise manipulation of MTHFR and related genes. Pooled CRISPR screens can identify modifiers of MTHFR dependency or drug sensitivity.
How CRISPR Can Be Used to Study GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity
Knockout
CRISPR knockout of MTHFR in cell lines such as HepG2 or HEK293 abolishes GO:0106313 activity, leading to elevated homocysteine and altered folate metabolism. These models are useful for studying the metabolic and phenotypic consequences of MTHFR loss.
Point Mutation
Introducing the C677T or A1298C point mutations via CRISPR base editing or HDR recreates common human variants in isogenic cell lines. These models help dissect the specific effects of reduced MTHFR activity on enzyme kinetics and downstream pathways.
Knock-in
Knock-in of tagged MTHFR (e.g., FLAG or GFP) allows for affinity purification and imaging of the enzyme in live cells. This approach can reveal subcellular localization and interaction partners.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of MTHFR can increase enzyme activity, providing a gain-of-function model to study the effects of enhanced folate metabolism on cell growth and methylation.
How EDITGENE Supports methylenetetrahydrofolate reductase (NADPH) activity Research
Researchers studying methylenetetrahydrofolate reductase (NADPH) activity-related genes often need to determine whether a candidate gene is causally involved in metabolic or disease phenotypes. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models, enabling functional validation of MTHFR and its network.
Contact EDITGENE today to design your custom CRISPR model for methylenetetrahydrofolate reductase (NADPH) activity research.
Frequently Asked Questions About methylenetetrahydrofolate reductase (NADPH) activity
What is GO:0106313?
GO:0106313 is the Gene Ontology term for methylenetetrahydrofolate reductase (NADPH) activity, the enzyme that converts 5-methyl-THF to 5,10-methylene-THF using NADP+.
What does MTHFR do in the body?
MTHFR catalyzes a key step in folate metabolism, helping to regulate homocysteine and produce methyl groups for DNA methylation and methionine synthesis.
What genes are involved in methylenetetrahydrofolate reductase (NADPH) activity?
The primary gene is MTHFR, but related genes include MTR, MTRR, CBS, and SHMT1, which interact in one-carbon metabolism.
How does the C677T mutation affect MTHFR activity?
The C677T polymorphism reduces MTHFR enzyme activity, leading to elevated homocysteine and increased risk of certain diseases.
Is MTHFR activity associated with disease?
Yes, altered MTHFR activity has been linked to cardiovascular disease, pregnancy complications, psychiatric disorders, and drug sensitivity in tuberculosis.
Can CRISPR be used to study MTHFR?
Yes, CRISPR knockout, knock-in, and point mutation models allow precise functional studies of MTHFR in cells.
What are the symptoms of MTHFR deficiency?
Severe MTHFR deficiency is rare and can cause neurological problems, but common variants may be asymptomatic or associated with mild hyperhomocysteinemia.
How is MTHFR activity measured?
MTHFR activity is typically measured in cell lysates using enzymatic assays that monitor NADPH oxidation or substrate conversion.
What is the role of NADPH in MTHFR activity?
NADPH is a cofactor; the reaction uses NADP+ as an electron acceptor, producing NADPH and H+.
Are there drugs that target MTHFR?
MTHFR is not a direct drug target in humans, but in Mycobacterium tuberculosis, reduced MTHFR activity increases sensitivity to para-aminosalicylic acid.
Conclusion
GO:0106313 methylenetetrahydrofolate reductase (NADPH) activity represents a fundamental biochemical function with far-reaching implications for human health and disease. From its role in one-carbon metabolism to its association with common polymorphisms and diverse clinical phenotypes, MTHFR continues to be a focus of genetic and metabolic research. Leveraging CRISPR-based models and EDITGENE's services can accelerate the discovery of novel insights and therapeutic strategies targeting this pathway.
References
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- 2. Araszkiewicz AF et al.. 2025. MTHFR Gene Polymorphisms: A Single Gene with Wide-Ranging Clinical Implications-A Review.. Genes (Basel) 16(4) PMID: 40282401
- 3. Weile J et al.. 2021. Shifting landscapes of human MTHFR missense-variant effects.. Am J Hum Genet 108(7):1283-1300 PMID: 34214447
- 4. Wan L et al.. 2018. Methylenetetrahydrofolate reductase and psychiatric diseases.. Transl Psychiatry 8(1):242 PMID: 30397195
- 5. Yu JF et al.. 2022. Decreased Methylenetetrahydrofolate Reductase Activity Leads to Increased Sensitivity to para-Aminosalicylic Acid in Mycobacterium tuberculosis.. Antimicrob Agents Chemother 66(1):e0146521 PMID: 34780266
- 6. Altwayan R et al.. 2025. Comprehensive review of thrombophilia: pathophysiology, prevalence, risk factors, and molecular diagnosis.. Transfus Clin Biol 32(2):228-244 PMID: 40157494
- 7. Mazloomi S et al.. 2020. Evaluation of methylenetetrahydrofolate reductase (MTHFR) activity and the levels of homocysteine and malondialdehyde (MDA) in the serum of women with preeclampsia.. Clin Exp Hypertens 42(7):590-594 PMID: 32174183
- 8. Tietjen GE et al.. 2018. Hypercoagulability and Migraine.. Headache 58(1):173-183 PMID: 28181217
