GO:0008705 methionine synthase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0008705 methionine synthase activity catalyzes the cobalamin-dependent transfer of a methyl group from (6S)-5-methyltetrahydrofolate to L-homocysteine, producing tetrahydrofolate and L-methionine.
• The enzyme is a modular cobalamin-dependent methyltransferase that cycles between methylcob(III)alamin and cob(I)alamin states, with reactivation by methionine synthase reductase.
• Loss of methionine synthase activity impairs folate cycling, causes hyperhomocysteinemia, and is linked to neural tube defects, liver disease, and neurological disorders.
• Tumour cells depend on methionine synthase to sustain tetrahydrofolate pools, making it a potential metabolic vulnerability in cancer.
• Methionine synthase activity is regulated by vitamin B12 availability, oxidative stress, nitric oxide, and heat-shock proteins.
• CRISPR knockout, point-mutation, knock-in, and overexpression models enable causal dissection of MTR and MTRR function in folate and methionine metabolism.
Description
Methionine synthase activity (GO:0008705) is a molecular function that sits at the intersection of folate and methionine metabolism. It catalyzes the reaction (6S)-5-methyl-5,6,7,8-tetrahydrofolate + L-homocysteine = (6S)-5,6,7,8-tetrahydrofolate + L-methionine, thereby regenerating tetrahydrofolate for nucleotide synthesis and producing methionine for protein synthesis and methylation reactions. The enzyme is cobalamin-dependent and is one of only two known mammalian enzymes that use vitamin B12 as a cofactor. Because it couples the folate cycle to the methionine cycle, its activity is essential for maintaining cellular methylation potential and redox balance. Researchers study this term to understand how vitamin B12 deficiency, oxidative stress, and genetic variants in MTR and MTRR contribute to hyperhomocysteinemia, neural tube defects, and cancer metabolism. The enzyme is also a target for mechanistic studies of cobalamin chemistry and for the development of metabolic inhibitors.
methionine synthase activity At A Glance
| GO ID | GO:0008705 |
|---|---|
| GO term | methionine synthase activity |
| Ontology | molecular_function |
| Synonym | 5-methyltetrahydrofolate--homocysteine S-methyltransferase activity; cobalamin-dependent methionine synthase activity; MetH; vitamin B12 methyltransferase activity |
| Major function | Catalyzes methyl transfer from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate |
| Cofactor | Cobalamin (vitamin B12) |
| Substrates | (6S)-5-methyl-5,6,7,8-tetrahydrofolate and L-homocysteine |
| Products | (6S)-5,6,7,8-tetrahydrofolate and L-methionine |
| Pathway context | Folate cycle and methionine cycle |
| Representative gene | MTR (methionine synthase); MTRR (methionine synthase reductase) |
What Is GO:0008705?
According to the Gene Ontology, methionine synthase activity (GO:0008705) is defined as the catalysis of the reaction: (6S)-5-methyl-5,6,7,8-tetrahydrofolate + L-homocysteine = (6S)-5,6,7,8-tetrahydrofolate + L-methionine. In other words, it is the cobalamin-dependent transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, yielding methionine and tetrahydrofolate. This activity is synonymous with 5-methyltetrahydrofolate--homocysteine S-methyltransferase activity, cobalamin-dependent methionine synthase activity, and MetH.
Why Is methionine synthase activity Important in Cell Biology?
Methionine synthase activity is critical for one-carbon metabolism, linking folate and methionine cycles to nucleotide synthesis, methylation, and redox homeostasis. Its dysfunction causes hyperhomocysteinemia, a risk factor for neural tube defects, cardiovascular disease, and neurological disorders. In cancer, methionine synthase supports tumour tetrahydrofolate pools and may be a metabolic target. Understanding its regulation by vitamin B12, oxidative stress, and nitric oxide is essential for interpreting nutritional and genetic studies.
• Maintains methionine and tetrahydrofolate pools for protein synthesis and nucleotide biosynthesis.
• Prevents homocysteine accumulation, a biomarker of cardiovascular and neurological risk.
• Supports methylation reactions via S-adenosylmethionine regeneration.
• Its impairment is linked to neural tube defects in fetuses.
• Tumour cells rely on methionine synthase for tetrahydrofolate pools, suggesting a cancer metabolic target.
• Regulated by vitamin B12 status, oxidative stress, and nitric oxide.
• Methionine synthase reductase (MTRR) maintains its activity by reductive reactivation.
• Heat-shock proteins and cobalamin availability influence its stability and function.
• Hepatic methionine synthase activity is altered by choline supply and energy balance in dairy cows, relevant to animal nutrition.
• Genetic variants in MTR and MTRR affect enzyme activity and disease susceptibility.
Molecular Mechanism of methionine synthase activity
Substrate binding and methyl transfer
In simple terms: The enzyme grabs a methyl group from one molecule and hands it to another.
Methionine synthase binds (6S)-5-methyl-5,6,7,8-tetrahydrofolate and L-homocysteine. The methyl group is transferred from 5-methyltetrahydrofolate to the cobalamin cofactor, forming methylcob(III)alamin, and then to homocysteine to produce methionine and tetrahydrofolate.
Cobalamin redox cycling
In simple terms: The vitamin B12 cofactor changes its oxidation state to accept and donate methyl groups.
The cobalamin cofactor cycles between methylcob(III)alamin and cob(I)alamin. The cob(I)alamin form is highly reactive and can be oxidized to cob(II)alamin, which inactivates the enzyme. Reactivation requires reductive methylation by methionine synthase reductase (MTRR).
Reactivation by methionine synthase reductase
In simple terms: A partner enzyme repairs methionine synthase when it gets stuck.
Methionine synthase reductase (MTRR) uses NADPH to reduce cob(II)alamin back to cob(I)alamin and then methylates it to methylcob(III)alamin, restoring methionine synthase activity. This reactivation is essential under oxidative stress.
Regulation by nitric oxide and oxidative stress
In simple terms: Stress molecules can shut down the enzyme.
Nitric oxide inhibits methionine synthase activity in vivo, disrupting carbon flow through the folate pathway. Oxidative stress can oxidize the cobalamin cofactor, leading to enzyme inactivation unless MTRR reactivates it.
Role of heat-shock proteins and cobalamin
In simple terms: Chaperones and vitamin B12 help keep the enzyme working.
Heat-shock proteins and cobalamin availability are important for maintaining methionine synthase activity. In conditions of cobalamin deficiency, enzyme activity decreases, contributing to impaired methionine synthesis.
Key Genes Involved in GO:0008705 methionine synthase activity
The following genes encode proteins directly involved in methionine synthase activity, its regulation, and related metabolic pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTR | Encodes methionine synthase, the enzyme catalyzing GO:0008705 | Mutations cause hyperhomocysteinemia and neural tube defects |
| MTRR | Encodes methionine synthase reductase, reactivates MTR | Defects lead to impaired methionine synthesis and disease |
| MTHFR | Encodes methylenetetrahydrofolate reductase, produces 5-methyltetrahydrofolate | Polymorphisms affect folate metabolism and MTR substrate supply |
| MTHFD1 | Encodes methylenetetrahydrofolate dehydrogenase, folate cycle | Supports one-carbon supply for methionine synthesis |
| BHMT | Encodes betaine-homocysteine methyltransferase, alternative homocysteine remethylation | Compensates for methionine synthase in liver |
| CBS | Encodes cystathionine beta-synthase, transsulfuration | Regulates homocysteine levels |
| MAT1A | Encodes methionine adenosyltransferase, produces SAM | Links methionine to methylation |
| GNMT | Encodes glycine N-methyltransferase, regulates SAM/SAH ratio | Affects methylation potential |
| AHCY | Encodes S-adenosylhomocysteine hydrolase | Controls homocysteine and methylation |
| SLC46A1 | Encodes proton-coupled folate transporter | Folate uptake affects MTR substrate |
| TCN2 | Encodes transcobalamin II, vitamin B12 transport | B12 delivery to cells affects MTR activity |
| CUBN | Encodes cubilin, B12 absorption | B12 uptake influences MTR |
| AMN | Encodes amnionless, B12 absorption | B12 uptake influences MTR |
| FUT2 | Encodes fucosyltransferase 2, B12 status modifier | Affects B12 availability |
| HSPA1A | Encodes heat-shock protein 70, maintains MTR activity | Chaperone support for MTR |
| HSPA8 | Encodes HSC70, chaperone | Maintains MTR activity |
| NOS2 | Encodes inducible nitric oxide synthase, produces NO | NO inhibits MTR |
| NOS3 | Encodes endothelial nitric oxide synthase | NO inhibits MTR |
How Is methionine synthase activity Regulated?
Methionine synthase activity is regulated at multiple levels. Vitamin B12 availability is a primary determinant; cobalamin deficiency reduces enzyme activity. Oxidative stress can oxidize the cobalamin cofactor, inactivating the enzyme, while methionine synthase reductase (MTRR) reactivates it using NADPH. Nitric oxide inhibits methionine synthase in vivo, disrupting folate metabolism. Heat-shock proteins and cobalamin help maintain activity under stress. In liver, choline supply and energy balance alter methionine synthase activity, reflecting nutritional regulation.
methionine synthase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTR | Neural tube defects, hyperhomocysteinemia | Mtr knockout mouse, patient-derived iPSCs |
| MTRR | Methionine synthase deficiency, neurological disease | Mtrr knockout cell lines, knock-in of patient mutations |
| MTHFR | Hyperhomocysteinemia, cardiovascular risk | Mthfr knockout mice, point-mutation models |
| BHMT | Liver disease, choline metabolism | Bhmt knockout mice, hepatic cell lines |
| NOS2 | Inflammation, nitric oxide-mediated inhibition | Nos2 knockout macrophages, overexpression models |
Neural tube defects and vitamin B12 deficiency
Impaired methionine synthase activity due to vitamin B12 deficiency or MTR mutations is associated with neural tube defects. Fetuses with neural tube defects show decreased liver methionine synthase activity and expression. Causes include inherited disorders of B12 metabolism and acquired deficiency.
Hyperhomocysteinemia and cardiovascular disease
Loss of methionine synthase activity leads to homocysteine accumulation, a risk factor for cardiovascular disease and stroke. Impaired enzyme activity is a consequence of B12 deficiency and genetic variants in MTR and MTRR.
Cancer metabolism
Methionine synthase supports tumour tetrahydrofolate pools, and its inhibition may impair cancer cell proliferation. This makes it a potential target for metabolic therapy.
Neurological and hepatic disorders
Methionine synthase dysfunction contributes to neurological disorders and liver disease. In dairy cows, hepatic methionine synthase activity is altered by choline supply during negative energy balance, linking nutrition to liver metabolism.
From methionine synthase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of MTR affect methionine synthesis and folate pools? | MTR knockout cell lines (e.g., HCT116, HEK293) |
| How do patient mutations in MTR affect enzyme activity? | Point-mutation knock-in via CRISPR in cell lines |
| Does MTRR reactivation require specific domains? | Domain-specific knock-in or knockout of MTRR |
| Can methionine synthase be tagged for localization studies? | Endogenous knock-in of fluorescent or epitope tags |
| Does overexpression of MTR rescue B12 deficiency phenotypes? | Overexpression cell models |
| What is the role of MTR in tumour growth? | Xenograft models with MTR knockout or overexpression |
How to Study the methionine synthase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzymatic assay | Methionine synthase activity | Tissue and cell lysates |
| LC-MS metabolomics | Folate and methionine cycle intermediates | Cell and tumour samples |
| Stable isotope tracing | Flux through methionine synthase | Cancer metabolism studies |
| Western blot | MTR and MTRR protein levels | Disease models |
| qRT-PCR | MTR and MTRR mRNA expression | Genetic and nutritional studies |
| CRISPR knockout screen | Genes required for methionine synthase function | Functional genomics |
| Bioinformatics pathway analysis | Metabolic network reconstruction | Data integration |
Enzymatic activity assays
Methionine synthase activity is measured using radiolabeled substrates or coupled assays that monitor methionine formation. These assays are used to quantify enzyme activity in tissues and cell lysates.
Metabolomics and flux analysis
Metabolomics quantifies folate and methionine cycle intermediates. Stable isotope tracing can measure flux through methionine synthase in cells and tumours.
Western blotting and immunodetection
Western blotting detects MTR and MTRR protein levels. This is used to correlate expression with activity in disease models.
CRISPR screening and functional genomics
CRISPR knockout screens can identify genes that modulate methionine synthase activity or synthetic lethality with MTR loss. Bioinformatics analysis of screening data reveals pathways and networks.
How CRISPR Can Be Used to Study GO:0008705 methionine synthase activity
Knockout
CRISPR knockout of MTR or MTRR eliminates methionine synthase activity, causing methionine auxotrophy and homocysteine accumulation. These models are used to study metabolic dependencies and compensatory pathways.
Point Mutation
Point mutations identified in patients with MTR deficiency can be introduced into cell lines to assess their impact on enzyme activity and stability. This helps classify variants of uncertain significance.
Knock-in
Knock-in of tagged MTR or MTRR allows visualization and immunoprecipitation of the endogenous proteins. This is useful for studying localization and interactors.
Overexpression
Overexpression of MTR or MTRR can rescue phenotypes associated with B12 deficiency or oxidative stress. It is also used to study gain-of-function effects in cancer models.
How EDITGENE Supports methionine synthase activity Research
Researchers studying methionine synthase activity-related genes often need to determine whether a candidate gene is causally involved in folate and methionine metabolism. EDITGENE provides CRISPR-based cell model services to enable such causal studies.
Contact EDITGENE today to design your custom CRISPR model for methionine synthase activity research.
Frequently Asked Questions About methionine synthase activity
What is methionine synthase activity?
Methionine synthase activity (GO:0008705) is the cobalamin-dependent catalysis of methyl transfer from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate.
What genes are involved in methionine synthase activity?
The main genes are MTR, encoding methionine synthase, and MTRR, encoding methionine synthase reductase, which reactivates the enzyme.
What is the role of vitamin B12 in methionine synthase activity?
Vitamin B12 (cobalamin) is the cofactor that accepts and transfers methyl groups in the reaction.
How is methionine synthase activity regulated?
It is regulated by vitamin B12 availability, oxidative stress, nitric oxide, and reactivation by methionine synthase reductase.
What diseases are associated with impaired methionine synthase activity?
Impaired activity is linked to neural tube defects, hyperhomocysteinemia, cardiovascular disease, and neurological disorders.
How can I measure methionine synthase activity?
Enzymatic assays using radiolabeled substrates or coupled assays measure activity in cell and tissue lysates.
What CRISPR models are available for studying methionine synthase?
Knockout, point-mutation, knock-in, and overexpression models can be generated for MTR and MTRR.
Does methionine synthase play a role in cancer?
Yes, methionine synthase supports tumour tetrahydrofolate pools and is a potential metabolic target.
What is the difference between MTR and MTRR?
MTR is the enzyme that catalyzes the reaction; MTRR is the reductase that reactivates MTR by reducing its cobalamin cofactor.
Can methionine synthase activity be inhibited by nitric oxide?
Yes, nitric oxide inhibits methionine synthase in vivo and disrupts folate metabolism.
Conclusion
Methionine synthase activity (GO:0008705) is a central molecular function that connects folate and methionine metabolism through a cobalamin-dependent methyl transfer reaction. Its regulation by vitamin B12, oxidative stress, and methionine synthase reductase is critical for health, and its dysfunction contributes to neural tube defects, hyperhomocysteinemia, and cancer metabolism. CRISPR-based models provide powerful tools to dissect the causal roles of MTR and MTRR in these processes.
References
- 1. Banerjee RV et al.. 1990. Cobalamin-dependent methionine synthase.. FASEB J 4(5):1450-9 PMID: 2407589
- 2. Grabowski M et al.. 2012. Role of heat-shock proteins and cobalamine in maintaining methionine synthase activity.. Acta Biochim Pol 59(4):489-93 PMID: 23251906
- 3. Guéant JL et al.. 2022. Causes and consequences of impaired methionine synthase activity in acquired and inherited disorders of vitamin B(12) metabolism.. Crit Rev Biochem Mol Biol 57(2):133-155 PMID: 34608838
- 4. Pabst FR et al.. 2025. Methionine synthase reductase regulates heterochromatin independently of methionine synthesis through mitochondrial homeostasis.. bioRxiv PMID: 41509232
- 5. Ghergurovich JM et al.. 2021. Methionine synthase supports tumour tetrahydrofolate pools.. Nat Metab 3(11):1512-1520 PMID: 34799699
- 6. Coleman DN et al.. 2019. Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows.. J Dairy Sci 102(9):8305-8318 PMID: 31301838
- 7. Fofou-Caillierez MB et al.. 2019. Vitamin B-12 and liver activity and expression of methionine synthase are decreased in fetuses with neural tube defects.. Am J Clin Nutr 109(3):674-683 PMID: 30848279
- 8. Danishpajooh IO et al.. 2001. Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway.. J Biol Chem 276(29):27296-303 PMID: 11371572
