GO:0030272 5-formyltetrahydrofolate cyclo-ligase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0030272 describes the ATP-dependent conversion of 5-formyltetrahydrofolate (5-FTHF) to 5,10-methenyltetrahydrofolate (5,10-MTHF), a reaction that recycles a stable folate metabolite back into one-carbon metabolism.
• The enzyme is a cyclo-ligase that uses ATP and releases ADP, inorganic phosphate and a proton, and it is structurally and mechanistically distinct from the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
• 5-FTHF is the only naturally occurring folate with a formyl group at the N5 position, and it is a potent inhibitor of several folate-dependent enzymes, so its removal by this activity is metabolically important.
• In plants, 5-formyltetrahydrofolate cyclo-ligase (5-FCL) is essential for normal folate homeostasis, and loss of the enzyme causes 5-FTHF accumulation and growth defects.
• In humans, the enzyme is encoded by MTHFS, and altered 5-FTHF levels have been linked to cerebral folate deficiency and to modulation of antifolate drug activity.
• CRISPR knockout, point-mutation, knock-in and overexpression models are powerful tools for dissecting the role of MTHFS and related one-carbon enzymes in health and disease.
Description
5-formyltetrahydrofolate cyclo-ligase activity (GO:0030272) is a molecular function that catalyzes the ATP-dependent cyclization of 5-formyltetrahydrofolate (5-FTHF) to 5,10-methenyltetrahydrofolate (5,10-MTHF), releasing ADP, phosphate and a proton. This reaction is part of the broader folate one-carbon network, which supplies methyl groups for nucleotide synthesis, amino acid metabolism and methylation reactions. The enzyme is widely distributed across bacteria, plants and mammals, and its bacterial and plant homologs have been structurally and biochemically characterized. Because 5-FTHF is a stable folate derivative that can inhibit several folate-dependent enzymes, its conversion by this cyclo-ligase is thought to be important for maintaining folate homeostasis and one-carbon flux. In humans, the enzyme is encoded by MTHFS, and its activity influences the cellular response to antifolate drugs such as pralatrexate and methotrexate. This article summarizes the definition, mechanism, key genes, disease links and research methods for GO:0030272, with a focus on how CRISPR-based models can be used to study this activity.
5-formyltetrahydrofolate cyclo-ligase activity At A Glance
| GO ID | GO:0030272 |
|---|---|
| GO term | 5-formyltetrahydrofolate cyclo-ligase activity |
| Ontology | molecular_function |
| Synonym | 5,10-methenyltetrahydrofolate synthetase activity; 5-formyltetrahydrofolate cyclodehydrase; methenyl-THF synthetase activity |
| Major function | ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate |
| Reaction | 5-formyltetrahydrofolate + ATP = 5,10-methenyltetrahydrofolate + ADP + H+ + phosphate |
| Cofactor | ATP (required); Mg2+ may be required for activity |
| Subcellular location | Cytoplasm (folate one-carbon metabolism) |
| Representative enzyme | MTHFS (human), 5-FCL (plant), BA4489 (Bacillus anthracis) |
What Is GO:0030272?
GO:0030272 is defined as the catalysis of the reaction: 5-formyltetrahydrofolate + ATP = 5,10-methenyltetrahydrofolate + ADP + H+ + phosphate. In other words, it is an ATP-dependent cyclo-ligase that closes a formyl group onto the pteridine ring of 5-FTHF to form the methenyl bridge of 5,10-MTHF, consuming one molecule of ATP and releasing ADP, inorganic phosphate and a proton. The reaction is reversible in principle, but under physiological conditions it is generally considered to drive the recycling of 5-FTHF back into the active one-carbon pool.
Why Is 5-formyltetrahydrofolate cyclo-ligase activity Important in Cell Biology?
GO:0030272 is important because it controls the cellular level of 5-formyltetrahydrofolate, a folate derivative that is chemically stable but can act as an inhibitor of several folate-dependent enzymes. By converting 5-FTHF to 5,10-methenyltetrahydrofolate, the enzyme recycles this metabolite into the one-carbon pool, supporting nucleotide biosynthesis, amino acid interconversion and methylation reactions. In plants, loss of 5-formyltetrahydrofolate cyclo-ligase leads to 5-FTHF accumulation and growth defects, demonstrating that the activity is required for normal folate homeostasis. In humans, the enzyme encoded by MTHFS modulates the anti-tumor activity of pralatrexate and methotrexate in HeLa cells, suggesting that it can influence the efficacy of antifolate chemotherapy. Moreover, 5-FTHF has been implicated in cerebral folate deficiency, a neurological condition that can be diagnosed by measuring folate metabolites in cerebrospinal fluid. Thus, understanding this activity is relevant to folate metabolism, cancer pharmacology and neurobiology.
• Recycles 5-formyltetrahydrofolate, the most stable folate derivative, back into the one-carbon pool.
• Supports nucleotide biosynthesis and amino acid metabolism by maintaining 5,10-methenyltetrahydrofolate availability.
• Modulates the cellular response to antifolate drugs such as pralatrexate and methotrexate.
• Loss of function in plants causes 5-FTHF accumulation and growth defects, highlighting its role in folate homeostasis.
• The enzyme is structurally distinct from other folate enzymes, making it a potential target for inhibitor design.
• 5-FTHF levels are relevant to cerebral folate deficiency and other neurological disorders.
• The bacterial enzyme from Bacillus anthracis has been crystallized, providing a structural template for drug discovery.
• The Mycoplasma pneumoniae enzyme has been used to probe the amino acid residues in the 5-FTHF binding site.
• The activity is conserved across bacteria, plants and mammals, making model organisms useful for functional studies.
• CRISPR-based knockout and knock-in models can help determine whether MTHFS is causally involved in disease phenotypes.
Molecular Mechanism of 5-formyltetrahydrofolate cyclo-ligase activity
Substrate binding and recognition
In simple terms: The enzyme first grabs its two starting materials, 5-formyltetrahydrofolate and ATP.
The enzyme binds 5-formyltetrahydrofolate (5-FTHF) and ATP in a sequential manner. Structural studies of the Bacillus anthracis enzyme BA4489 have revealed a fold that accommodates the pteridine ring of 5-FTHF and the nucleotide in adjacent pockets. Mutational analysis of the Mycoplasma pneumoniae enzyme has identified specific amino acid residues in the 5-FTHF binding site that are important for substrate recognition and catalysis. The binding of 5-FTHF is thought to position the formyl group for attack by the N10 nitrogen of the pteridine ring.
ATP-dependent activation and cyclization
In simple terms: ATP provides energy to close the formyl group onto the ring, forming a new bridge.
The reaction proceeds via an ATP-dependent activation step. The enzyme catalyzes the formation of a phosphoanhydride intermediate or a similar activated species, which then undergoes intramolecular cyclization to form the methenyl bridge of 5,10-methenyltetrahydrofolate (5,10-MTHF). This step releases ADP and inorganic phosphate. The overall reaction is: 5-formyltetrahydrofolate + ATP = 5,10-methenyltetrahydrofolate + ADP + H+ + phosphate. The enzyme is therefore classified as a cyclo-ligase, because it forms a new ring by ligating two atoms within the same molecule using ATP.
Product release and reversibility
In simple terms: After the new ring is made, the product is released and can be used elsewhere.
Following cyclization, 5,10-MTHF is released from the active site. This product is a central metabolite in one-carbon metabolism, where it can be converted to other folate derivatives such as 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate. The reaction is reversible in vitro, but the physiological direction is likely toward 5,10-MTHF formation, because 5-FTHF is a stable metabolite that would otherwise accumulate. In plants, the enzyme is known as 5-formyltetrahydrofolate cycloligase (5-FCL), and its activity is essential for preventing 5-FTHF accumulation.
Inhibition and regulation
In simple terms: The enzyme can be slowed down by certain molecules, and its activity is tuned to the cell's needs.
The activity of 5-formyltetrahydrofolate cyclo-ligase can be inhibited by substrate analogs and by products of folate metabolism. For example, inhibition of 5,10-methenyltetrahydrofolate synthetase has been studied as a way to probe the enzyme's role in one-carbon metabolism. In plants, 5-FTHF itself is an inhibitory but well-tolerated metabolite, and its accumulation in 5-FCL mutants suggests feedback regulation. In humans, the enzyme encoded by MTHFS is sensitive to antifolate drugs, and its activity can influence the cellular response to pralatrexate and methotrexate. The expression of MTHFS may also be regulated at the transcriptional level in response to folate status, although the exact mechanisms are not fully defined.
Key Genes Involved in GO:0030272 5-formyltetrahydrofolate cyclo-ligase activity
The following genes and proteins are directly or indirectly involved in 5-formyltetrahydrofolate cyclo-ligase activity and its metabolic context.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTHFS | Human 5-formyltetrahydrofolate cyclo-ligase; converts 5-FTHF to 5,10-MTHF | Target for studying folate metabolism and antifolate drug response |
| MTHFD1 | Bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase; interconverts folate derivatives | Provides 5,10-MTHF for downstream reactions |
| MTHFD2 | Mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase | Supports one-carbon metabolism in proliferating cells |
| MTHFR | Methylenetetrahydrofolate reductase; produces 5-methyltetrahydrofolate | Links folate cycle to methionine synthesis |
| SHMT1 | Serine hydroxymethyltransferase; generates 5,10-methylene-THF | Provides one-carbon units for folate metabolism |
| SHMT2 | Mitochondrial serine hydroxymethyltransferase | Supports mitochondrial one-carbon flux |
| GART | Phosphoribosylglycinamide formyltransferase; uses 10-formyl-THF | Connects folate metabolism to purine synthesis |
| ATIC | AICAR transformylase/IMP cyclohydrolase; uses 10-formyl-THF | Links folate cycle to purine biosynthesis |
| TYMS | Thymidylate synthase; uses 5,10-methylene-THF | Connects folate metabolism to DNA synthesis |
| DHFR | Dihydrofolate reductase; regenerates tetrahydrofolate | Target of methotrexate and other antifolates |
| FPGS | Folylpolyglutamate synthetase; adds glutamate tails to folates | Determines cellular retention of folates |
| GGH | Gamma-glutamyl hydrolase; removes glutamate tails | Modulates folate polyglutamation |
| SLC19A1 | Reduced folate carrier; transports folates into cells | Influences intracellular folate levels |
| FOLR1 | Folate receptor alpha; mediates folate uptake | Relevant to cerebral folate deficiency |
| BA4489 | Bacillus anthracis 5-formyltetrahydrofolate cyclo-ligase | Structural model for enzyme mechanism |
| 5-FCL | Arabidopsis thaliana 5-formyltetrahydrofolate cycloligase | Plant model for folate homeostasis |
| Mpn-MTHFS | Mycoplasma pneumoniae 5,10-methenyltetrahydrofolate synthetase | Used to study substrate binding residues |
How Is 5-formyltetrahydrofolate cyclo-ligase activity Regulated?
The activity of 5-formyltetrahydrofolate cyclo-ligase is regulated at multiple levels. In plants, the enzyme is known as 5-FCL, and its loss leads to 5-FTHF accumulation, suggesting that the enzyme is part of a feedback loop that maintains folate homeostasis. In humans, MTHFS expression may be influenced by folate status and by the availability of one-carbon donors, although the precise transcriptional and post-transcriptional mechanisms are not fully defined. The enzyme's activity can also be modulated by its substrate and product levels, as 5-FTHF is an inhibitory metabolite that can affect other folate-dependent enzymes. Additionally, antifolate drugs such as pralatrexate and methotrexate can alter the flux through this reaction by changing the cellular folate pool.
5-formyltetrahydrofolate cyclo-ligase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTHFS | Cerebral folate deficiency; antifolate drug response | MTHFS knockout HeLa cells; patient-derived fibroblasts |
| MTHFR | Hyperhomocysteinemia; neural tube defects | MTHFR knockout mice; iPSC-derived neurons |
| DHFR | Methotrexate resistance; cancer | DHFR knockout cancer cell lines; xenografts |
| FOLR1 | Cerebral folate deficiency | FOLR1 knockout mice; patient iPSC-derived choroid plexus cells |
| SLC19A1 | Folate transport defects | SLC19A1 knockout cell lines; intestinal organoids |
Cerebral folate deficiency
Cerebral folate deficiency is a neurological disorder characterized by low levels of 5-methyltetrahydrofolate in the cerebrospinal fluid despite normal serum folate. Although the primary cause is often autoantibodies against the folate receptor, abnormalities in folate metabolism, including 5-FTHF handling, can contribute to the condition. Measurement of folate metabolites in cerebrospinal fluid is part of the diagnostic workup, and the enzyme 5-formyltetrahydrofolate cyclo-ligase may influence the levels of these metabolites.
Cancer and antifolate chemotherapy
5-FTHF can modulate the anti-tumor activity of antifolate drugs. In HeLa cells, the presence of 5-FTHF affected the cytotoxicity of pralatrexate and methotrexate, suggesting that the enzyme that consumes 5-FTHF, namely 5-formyltetrahydrofolate cyclo-ligase, can influence drug response. Therefore, MTHFS expression levels or activity may serve as a biomarker for antifolate sensitivity, and targeting this enzyme could be a strategy to enhance chemotherapy efficacy.
Folate-related metabolic disorders
Inborn errors of folate metabolism can affect one-carbon flux and lead to developmental and neurological symptoms. Although mutations in MTHFS have not been widely reported, the enzyme is part of the folate pathway, and its dysfunction could contribute to 5-FTHF accumulation. In plants, loss of 5-FCL causes growth defects, indicating that the activity is essential for normal folate homeostasis. These findings suggest that human MTHFS deficiency could have metabolic consequences, but further studies are needed.
From 5-formyltetrahydrofolate cyclo-ligase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does MTHFS loss alter 5-FTHF levels and one-carbon flux? | MTHFS knockout HEK293 or HeLa cells |
| Does a point mutation in the active site abolish enzyme activity? | MTHFS point-mutation knock-in cells (e.g., catalytic residue substitution) |
| Does tagging MTHFS affect its localization or interactions? | MTHFS knock-in with FLAG or GFP tag |
| Does MTHFS overexpression change antifolate sensitivity? | MTHFS overexpression in cancer cell lines |
| Which genes interact with MTHFS in folate metabolism? | CRISPR library screening with folate-related sgRNA libraries |
| Does MTHFS loss affect neuronal folate handling? | MTHFS knockout iPSC-derived neurons |
How to Study the 5-formyltetrahydrofolate cyclo-ligase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | Conversion of 5-FTHF to 5,10-MTHF | Kinetic characterization and inhibitor testing |
| X-ray crystallography | Three-dimensional structure of the enzyme | Active site mapping and drug design |
| Site-directed mutagenesis | Effect of specific amino acid changes on activity | Identification of catalytic residues |
| LC-MS metabolomics | Intracellular folate species levels | Assessing 5-FTHF accumulation in mutants |
| CRISPR knockout | Loss-of-function phenotype | Determining gene essentiality and drug response |
| CRISPR activation | Gain-of-function phenotype | Identifying genes that rescue 5-FTHF toxicity |
| RNA-seq | Transcriptional changes upon perturbation | Mapping compensatory pathways |
| Proteomics | Protein expression and interactions | Identifying binding partners of MTHFS |
Enzymatic assays
The activity of 5-formyltetrahydrofolate cyclo-ligase can be measured spectrophotometrically by monitoring the formation of 5,10-methenyltetrahydrofolate at 350 nm or by coupling the reaction to NADP+ reduction. These assays are useful for determining kinetic parameters and for testing inhibitors. For example, inhibition of 5,10-methenyltetrahydrofolate synthetase has been studied using such assays.
Structural biology
X-ray crystallography and NMR can provide atomic-level insights into substrate binding and catalysis. The structure of the Bacillus anthracis enzyme BA4489 has been solved, revealing the overall fold and the active site architecture. Mutagenesis studies of the Mycoplasma pneumoniae enzyme have identified key residues in the 5-FTHF binding site. These methods are essential for understanding the mechanism and for structure-based drug design.
Metabolomics and folate profiling
Liquid chromatography-mass spectrometry (LC-MS) can quantify folate species, including 5-FTHF and 5,10-MTHF, in cells and tissues. This approach is used to assess the impact of enzyme knockout or overexpression on folate pools. In plants, folate profiling has been used to show that 5-FCL mutants accumulate 5-FTHF.
CRISPR-based genetic screens
Genome-wide CRISPR knockout or activation screens can identify genes that modulate sensitivity to antifolates or that regulate folate metabolism. Such screens can uncover synthetic lethal interactions with MTHFS loss and reveal pathways that compensate for reduced cyclo-ligase activity. These methods are powerful for discovering new therapeutic targets in folate-related diseases.
How CRISPR Can Be Used to Study GO:0030272 5-formyltetrahydrofolate cyclo-ligase activity
Knockout
CRISPR knockout of MTHFS can be used to eliminate 5-formyltetrahydrofolate cyclo-ligase activity and study its consequences on folate metabolism and cell growth. Knockout cells are expected to accumulate 5-FTHF, similar to plant 5-FCL mutants. These models can be used to test whether loss of MTHFS sensitizes cells to antifolate drugs or alters one-carbon flux.
Point Mutation
Point mutations in the MTHFS active site can be introduced to dissect the catalytic mechanism. For example, substituting residues identified in the Mycoplasma pneumoniae enzyme or the Bacillus anthracis structure can abolish activity without affecting protein stability. Such knock-in models allow precise structure-function studies in a physiological context.
Knock-in
Knock-in of a tagged version of MTHFS (e.g., FLAG or GFP) enables localization and interaction studies. Tagged knock-in cells can be used for immunoprecipitation and mass spectrometry to identify binding partners. Additionally, knock-in of disease-associated variants, if any, can help determine their functional impact.
Overexpression
Overexpression of MTHFS can increase the conversion of 5-FTHF to 5,10-MTHF and may alter cellular sensitivity to antifolates. Overexpression models are useful for testing whether increased enzyme activity can rescue phenotypes associated with 5-FTHF accumulation or enhance drug resistance.
How EDITGENE Supports 5-formyltetrahydrofolate cyclo-ligase activity Research
Researchers studying 5-formyltetrahydrofolate cyclo-ligase activity-related genes often need to determine whether a candidate gene is causally involved in a metabolic or disease phenotype. EDITGENE provides a comprehensive suite of CRISPR-based services to generate precisely engineered cell models, enabling functional validation of genes such as MTHFS and its partners.
Contact EDITGENE today to design your custom CRISPR model for 5-formyltetrahydrofolate cyclo-ligase activity research.
Frequently Asked Questions About 5-formyltetrahydrofolate cyclo-ligase activity
What is 5-formyltetrahydrofolate cyclo-ligase activity?
It is an enzymatic activity (GO:0030272) that converts 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate using ATP, releasing ADP, phosphate and a proton.
What is the reaction catalyzed by GO:0030272?
The reaction is: 5-formyltetrahydrofolate + ATP = 5,10-methenyltetrahydrofolate + ADP + H+ + phosphate.
Which gene encodes 5-formyltetrahydrofolate cyclo-ligase in humans?
In humans, the enzyme is encoded by MTHFS (5,10-methenyltetrahydrofolate synthetase).
What is the role of 5-formyltetrahydrofolate in the cell?
5-FTHF is a stable folate derivative that can inhibit folate-dependent enzymes; its conversion to 5,10-MTHF by this enzyme helps maintain one-carbon metabolism.
How is 5-formyltetrahydrofolate cyclo-ligase activity measured?
It can be measured spectrophotometrically by monitoring the formation of 5,10-methenyltetrahydrofolate at 350 nm or by coupled enzymatic assays.
What diseases are associated with 5-formyltetrahydrofolate cyclo-ligase dysfunction?
Altered 5-FTHF levels have been linked to cerebral folate deficiency and to modulation of antifolate drug activity in cancer.
What model organisms are used to study this enzyme?
Bacteria such as Bacillus anthracis and Mycoplasma pneumoniae, plants such as Arabidopsis thaliana, and mammalian cell lines are commonly used.
Can CRISPR be used to study 5-formyltetrahydrofolate cyclo-ligase?
Yes, CRISPR knockout, point mutation, knock-in and overexpression models can be used to dissect the function of MTHFS and its role in folate metabolism.
What is the difference between 5-formyltetrahydrofolate cyclo-ligase and 5,10-methenyltetrahydrofolate synthetase?
They refer to the same activity; 5,10-methenyltetrahydrofolate synthetase is a synonym for GO:0030272.
Why is 5-formyltetrahydrofolate cyclo-ligase important for drug discovery?
Because it controls 5-FTHF levels, which can modulate the efficacy of antifolate drugs like pralatrexate and methotrexate, making it a potential target for combination therapy.
Conclusion
5-formyltetrahydrofolate cyclo-ligase activity (GO:0030272) is a key enzymatic step in folate one-carbon metabolism that recycles 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Its importance spans plant folate homeostasis, bacterial metabolism and human health, where it influences antifolate drug response and possibly neurological conditions. The enzyme is structurally and mechanistically distinct, and its activity can be studied using enzymatic assays, structural biology, metabolomics and CRISPR-based genetic models. Future research using precise CRISPR engineering will help clarify the causal role of MTHFS in disease and identify new therapeutic opportunities.
References
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- 3. Field MS et al.. 2007. Inhibition of 5,10-methenyltetrahydrofolate synthetase.. Arch Biochem Biophys 458(2):194-201 PMID: 17250800
- 4. Pope S et al.. 2019. Cerebral folate deficiency: Analytical tests and differential diagnosis.. J Inherit Metab Dis 42(4):655-672 PMID: 30916789
- 5. Baggott JE et al.. 2003. 5,10-methenyltetrahydrofolate cyclohydrolase, rat liver and chemically catalysed formation of 5-formyltetrahydrofolate.. Biochem J 374(Pt 3):773-8 PMID: 12793858
- 6. Cooper C et al.. 2019. Investigations of Amino Acids in the 5-Formyltetrahydrofolate Binding Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumonia.. Protein J 38(4):409-418 PMID: 31401777
- 7. Goyer A et al.. 2005. 5-Formyltetrahydrofolate is an inhibitory but well tolerated metabolite in Arabidopsis leaves.. J Biol Chem 280(28):26137-42 PMID: 15888445
- 8. Jeanguenin L et al.. 2010. Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase.. J Biol Chem 285(53):41557-66 PMID: 20952389
