GO:0030586 [methionine synthase] reductase (NADPH) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0030586 describes the NADPH-dependent reductive reactivation of methionine synthase (MTR), converting inactive cob(II)alamin to active methylcob(I)alamin.
• The enzyme responsible, methionine synthase reductase (MTRR), is a dual-flavoprotein that transfers electrons from NADPH via FAD and FMN to MTR.
• MTRR also acts as a molecular chaperone for MTR, ensuring proper folding and stability.
• The MTRR A66G polymorphism (Ile22Met) is associated with increased risk of neural tube defects and several cancers.
• MTRR deficiency in mice causes hyperhomocysteinemia and metabolic derangements in folate and methionine cycles.
• Studying GO:0030586 requires tools like CRISPR knockout, point mutation, and biochemical assays to dissect its role in one-carbon metabolism.
Description
Methionine synthase (MTR) is a key enzyme in one-carbon metabolism that catalyzes the remethylation of homocysteine to methionine. During its catalytic cycle, the cobalamin cofactor can become oxidized to an inactive cob(II)alamin state, halting enzyme activity. The reactivation of MTR is carried out by methionine synthase reductase (MTRR), an NADPH-dependent diflavin oxidoreductase. This activity is formally described by the Gene Ontology term GO:0030586: [methionine synthase] reductase (NADPH) activity. Understanding this term is crucial for researchers studying folate metabolism, homocysteine regulation, and related diseases such as neural tube defects and cancer. The reaction catalyzed by MTRR maintains the pool of active MTR, thereby sustaining methionine synthesis and downstream methylation reactions.
[methionine synthase] reductase (NADPH) activity At A Glance
| GO ID | GO:0030586 |
|---|---|
| GO term | [methionine synthase] reductase (NADPH) activity |
| Ontology | molecular_function |
| Synonym | methionine synthase reductase activity; 5-methyltetrahydrofolate-homocysteine methyltransferase reductase; methionine synthase-cobalamin methyltransferase (cob(II)alamin reducing) |
| Major function | Reductive reactivation of methionine synthase (MTR) via NADPH-dependent electron transfer |
| Cofactors | FAD, FMN (in MTRR); cobalamin (in MTR) |
| Substrates | NADPH, H+, S-adenosyl methionine, [methionine synthase]-cob(II)alamin |
| Products | NADP+, S-adenosylhomocysteine, [methionine synthase]-methylcob(I)alamin |
| Related genes | MTRR (methionine synthase reductase), MTR (methionine synthase) |
What Is GO:0030586?
GO:0030586 defines the catalytic activity of an enzyme that reduces the oxidized form of methionine synthase (MTR) using NADPH as an electron donor. Specifically, it catalyzes the reaction: [methionine synthase]-cob(II)alamin + NADPH + H+ + S-adenosyl methionine = [methionine synthase]-methylcob(I)alamin + S-adenosylhomocysteine + NADP+. This reductive methylation restores MTR to its active methylcob(I)alamin state, enabling continued catalysis of homocysteine remethylation.
Why Is [methionine synthase] reductase (NADPH) activity Important in Cell Biology?
GO:0030586 is essential for maintaining methionine synthase activity, which is central to methionine synthesis, folate cycling, and methylation reactions. Dysregulation of this activity leads to elevated homocysteine, a risk factor for cardiovascular disease, neural tube defects, and cancer. Moreover, MTRR polymorphisms have been linked to tumor susceptibility and developmental anomalies, making this term a focus for genetic and clinical research.
• Maintains methionine synthase in its active state, supporting methionine and S-adenosylmethionine (SAM) production.
• Prevents hyperhomocysteinemia, a risk factor for vascular and neurological disorders.
• MTRR A66G polymorphism is associated with increased risk of neural tube defects.
• The same polymorphism contributes to susceptibility to multiple cancers.
• MTRR interacts with MMACHC and MMADHC, linking it to cobalamin trafficking disorders.
• MTRR acts as a chaperone for MTR, ensuring proper folding and function.
• Deficiency in MTRR causes metabolic derangement in mice, affecting folate and methionine cycles.
• The reaction mechanism involves electron transfer from NADPH via FAD and FMN, studied by stopped-flow spectrophotometry.
• Understanding GO:0030586 aids in designing therapies for homocysteine-related diseases.
• CRISPR-based models of MTRR mutations can elucidate disease mechanisms and drug responses.
Molecular Mechanism of [methionine synthase] reductase (NADPH) activity
Substrate Binding and Electron Transfer
In simple terms: MTRR grabs electrons from NADPH and delivers them to methionine synthase to switch it back on.
MTRR binds NADPH and transfers electrons to its FAD cofactor, then to FMN, and finally to the cob(II)alamin form of MTR. This electron transfer chain reduces cob(II)alamin to cob(I)alamin, which is then methylated by S-adenosylmethionine to form methylcob(I)alamin, reactivating MTR.
Reductive Methylation of MTR
In simple terms: The enzyme adds a methyl group to the reduced cobalamin, turning methionine synthase back to its working form.
Following reduction, MTRR facilitates the transfer of a methyl group from S-adenosylmethionine to cob(I)alamin, generating methylcob(I)alamin and S-adenosylhomocysteine. This step is essential for restoring the catalytic cycle of MTR.
Chaperone Function of MTRR
In simple terms: MTRR also helps methionine synthase fold correctly and stay stable.
Beyond its reductase activity, MTRR acts as a molecular chaperone for MTR, assisting in its folding and preventing aggregation. This dual function ensures that MTR remains functional even under stress conditions.
Regulation by NADPH and Redox State
In simple terms: The reaction depends on the availability of NADPH and the cell's redox balance.
MTRR activity is dependent on NADPH supply and is sensitive to oxidative stress. The enzyme's dual flavin cofactors allow it to shuttle electrons efficiently, but excess oxidation can impair its function, linking GO:0030586 to cellular redox regulation.
Key Genes Involved in GO:0030586 [methionine synthase] reductase (NADPH) activity
The following genes and proteins are directly involved in or regulate GO:0030586.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTRR | Methionine synthase reductase; catalyzes NADPH-dependent reduction of MTR | Polymorphisms linked to cancer and neural tube defects; target for functional studies |
| MTR | Methionine synthase; substrate of MTRR | Central to one-carbon metabolism; mutations cause homocystinuria |
| MMACHC | Cobalamin trafficking protein; interacts with MTRR | Mutations cause methylmalonic aciduria and homocystinuria |
| MMADHC | Cobalamin trafficking protein; interacts with MTRR | Defects lead to cobalamin disorders |
| MTHFR | Methylenetetrahydrofolate reductase; provides methyl groups for MTR | Polymorphisms affect folate metabolism and homocysteine levels |
| MTRR (A66G) | Common polymorphism (Ile22Met) in MTRR | Associated with increased risk of spina bifida and cancers |
| CBS | Cystathionine beta-synthase; transsulfuration pathway | Interacts with methionine cycle; nitrite reductase activity |
| MAT1A | Methionine adenosyltransferase; produces SAM | SAM is required for MTR reactivation |
| GNMT | Glycine N-methyltransferase; uses SAM | Regulates SAM/SAH ratio, affecting MTR activity |
| AHCY | S-adenosylhomocysteine hydrolase; removes SAH | SAH inhibits MTR reactivation; AHCY maintains flux |
| FAD | Flavin adenine dinucleotide; cofactor in MTRR | Essential for electron transfer |
| FMN | Flavin mononucleotide; cofactor in MTRR | Essential for electron transfer |
| NADPH | Electron donor for MTRR | Cellular redox status affects activity |
| S-adenosylmethionine | Methyl donor for MTR reactivation | Links methionine cycle to methylation reactions |
| S-adenosylhomocysteine | Product of MTR reactivation; inhibitor | Its accumulation inhibits MTR |
| MTRR (chaperone) | Chaperone function of MTRR | Ensures MTR stability |
| MTR (cob(II)alamin) | Oxidized form of MTR; substrate for MTRR | Accumulates under oxidative stress |
How Is [methionine synthase] reductase (NADPH) activity Regulated?
MTRR activity is regulated at multiple levels. Transcriptionally, the MTRR gene can be influenced by folate status and oxidative stress. Post-translationally, MTRR activity depends on NADPH availability and the redox state of the cell. Additionally, the interaction with MMACHC and MMADHC suggests that cobalamin trafficking regulates MTRR function. The A66G polymorphism alters enzyme efficiency and is a common genetic modifier.
[methionine synthase] reductase (NADPH) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTRR | Neural tube defects (spina bifida) | Mtrr knockout mouse; CRISPR point mutation (A66G) in cell lines |
| MTRR | Cancer susceptibility | MTRR knockout cancer cell lines; xenograft models |
| MTR | Homocystinuria | MTR knockout cells; patient-derived fibroblasts |
| MMACHC | Methylmalonic aciduria and homocystinuria | MMACHC knockout cell models; zebrafish |
| MMADHC | Cobalamin disorders | MMADHC knockout cells; mouse models |
Neural Tube Defects
The MTRR A66G polymorphism has been identified as a maternal risk factor for spina bifida, a neural tube defect. Reduced MTRR activity leads to impaired methionine synthase function, causing hyperhomocysteinemia and disrupted folate metabolism, which are implicated in neural tube closure defects.
Cancer Susceptibility
Meta-analyses have shown that the MTRR A66G polymorphism contributes to tumor susceptibility across various cancer types. Altered MTRR activity may affect DNA methylation and nucleotide synthesis, promoting carcinogenesis.
Cobalamin Disorders
MTRR interacts with MMACHC and MMADHC, proteins involved in intracellular cobalamin trafficking. Mutations in these proteins cause disorders like methylmalonic aciduria and homocystinuria, highlighting the importance of MTRR in cobalamin metabolism.
Hyperhomocysteinemia and Metabolic Derangement
Mice deficient in MTRR exhibit hyperhomocysteinemia and metabolic derangements in methionine and folate cycles, demonstrating the critical role of MTRR in maintaining normal metabolism.
From [methionine synthase] reductase (NADPH) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does MTRR loss affect methionine synthase activity? | MTRR knockout cell lines (e.g., HEK293) |
| Does the A66G polymorphism alter MTRR function? | Point mutation knock-in of MTRR A66G in cell lines |
| Can MTRR chaperone function be separated from reductase activity? | Knock-in of chaperone-deficient MTRR mutants |
| How does MTRR deficiency affect global methylation? | MTRR knockout mice; SAM/SAH quantification |
| Does MTRR interact with MMACHC/MMADHC in vivo? | Tagged knock-in of MTRR (e.g., GFP) for co-IP |
| Can overexpression of MTRR rescue MTR defects? | Overexpression of MTRR in MTR mutant cells |
How to Study the [methionine synthase] reductase (NADPH) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| NADPH oxidation assay | MTRR reductase activity | Enzyme kinetics and inhibitor screening |
| Stopped-flow spectrophotometry | Electron transfer rates | Mechanistic studies of flavin reduction |
| Genotyping (PCR-RFLP) | MTRR A66G polymorphism | Association studies in disease cohorts |
| Co-immunoprecipitation | Protein-protein interactions | Identifying MTRR complexes |
| LC-MS/MS | Metabolite levels (homocysteine, SAM, SAH) | Metabolic profiling in knockout models |
| Western blot | MTR and MTRR protein levels | Assessing expression and stability |
| CRISPR knockout | Gene function | Creating MTRR-deficient cell lines |
| RNA-seq | Transcriptional changes | Global effects of MTRR loss |
Biochemical Assays for MTRR Activity
MTRR activity can be measured spectrophotometrically by monitoring NADPH oxidation at 340 nm or by using stopped-flow spectrophotometry to track electron transfer to MTR.
Genetic and Epigenetic Analysis
Genotyping of the MTRR A66G polymorphism is performed by PCR-RFLP or TaqMan assays. Epigenetic studies can assess DNA methylation changes in MTRR knockout models.
Proteomic and Interaction Studies
Co-immunoprecipitation and mass spectrometry can identify MTRR interaction partners such as MMACHC and MMADHC. Tagged knock-in models facilitate these studies.
Metabolic Profiling
LC-MS/MS can quantify methionine, homocysteine, SAM, and SAH levels in cells or tissues to assess the impact of MTRR mutations on one-carbon metabolism.
How CRISPR Can Be Used to Study GO:0030586 [methionine synthase] reductase (NADPH) activity
Knockout
CRISPR knockout of MTRR in cell lines (e.g., HEK293, HeLa) can abolish reductase activity, leading to impaired methionine synthase function and elevated homocysteine. These models are useful for studying metabolic consequences and drug responses.
Point Mutation
Introducing the A66G point mutation into the MTRR gene via CRISPR base editing or HDR allows researchers to study its effect on enzyme activity and its association with neural tube defects and cancer.
Knock-in
Knock-in of tagged MTRR (e.g., GFP or FLAG) enables live-cell imaging and co-immunoprecipitation to study its localization and interactions with MTR, MMACHC, and MMADHC.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of MTRR can rescue phenotypes in MTR-deficient cells and test whether increased MTRR activity protects against hyperhomocysteinemia.
How EDITGENE Supports [methionine synthase] reductase (NADPH) activity Research
Researchers studying [methionine synthase] reductase (NADPH) activity-related genes often need to determine whether a candidate gene is causally involved in metabolic or disease phenotypes. EDITGENE provides comprehensive CRISPR-based services to create precise cellular models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for [methionine synthase] reductase (NADPH) activity research.
Frequently Asked Questions About [methionine synthase] reductase (NADPH) activity
What is GO:0030586?
GO:0030586 is a Gene Ontology molecular function term describing the NADPH-dependent reduction of methionine synthase (MTR) by methionine synthase reductase (MTRR), reactivating MTR's catalytic cycle.
What is methionine synthase reductase?
Methionine synthase reductase (MTRR) is a dual-flavoprotein enzyme that transfers electrons from NADPH to methionine synthase, restoring its active state.
What genes are involved in [methionine synthase] reductase (NADPH) activity?
The primary genes are MTRR (encoding the reductase) and MTR (encoding methionine synthase). Other interacting genes include MMACHC and MMADHC.
What diseases are associated with MTRR mutations?
MTRR mutations, particularly A66G, are linked to neural tube defects, cancer susceptibility, and hyperhomocysteinemia.
How is MTRR activity measured?
MTRR activity is typically measured by NADPH oxidation assays or stopped-flow spectrophotometry to monitor electron transfer.
What is the role of MTRR in folate metabolism?
MTRR maintains methionine synthase activity, which is essential for converting homocysteine to methionine and for folate cycling.
Can CRISPR be used to study MTRR function?
Yes, CRISPR knockout, point mutation, and knock-in models allow precise dissection of MTRR's role in metabolism and disease.
What is the A66G polymorphism in MTRR?
A66G is a common single nucleotide polymorphism in MTRR that results in an isoleucine-to-methionine substitution at position 22, associated with reduced enzyme activity and increased disease risk.
How does MTRR interact with methionine synthase?
MTRR physically interacts with MTR, transferring electrons to reduce the oxidized cobalamin cofactor and also acting as a chaperone.
What model systems are used to study MTRR deficiency?
Mouse models (Mtrr knockout) and CRISPR-edited cell lines are commonly used to study metabolic and developmental consequences.
Conclusion
GO:0030586, [methionine synthase] reductase (NADPH) activity, is a critical molecular function that sustains methionine synthase and one-carbon metabolism. Its dysregulation is implicated in neural tube defects, cancer, and hyperhomocysteinemia. Advanced CRISPR tools enable precise modeling of MTRR variants, offering insights into disease mechanisms and potential therapies.
References
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- 2. Olteanu H et al.. 2001. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation.. J Biol Chem 276(38):35558-63 PMID: 11466310
- 3. Wolthers KR et al.. 2004. Electron transfer in human methionine synthase reductase studied by stopped-flow spectrophotometry.. Biochemistry 43(2):490-500 PMID: 14717604
- 4. Yamada K et al.. 2006. Human methionine synthase reductase is a molecular chaperone for human methionine synthase.. Proc Natl Acad Sci U S A 103(25):9476-81 PMID: 16769880
- 5. Han D et al.. 2012. Methionine synthase reductase A66G polymorphism contributes to tumor susceptibility: evidence from 35 case-control studies.. Mol Biol Rep 39(2):805-16 PMID: 21547363
- 6. Elmore CL et al.. 2007. Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase.. Mol Genet Metab 91(1):85-97 PMID: 17369066
- 7. Gherasim C et al.. 2014. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.. PLoS One 9(1):e85544 PMID: 24416422
- 8. van der Linden IJ et al.. 2006. The methionine synthase reductase 66A>G polymorphism is a maternal risk factor for spina bifida.. J Mol Med (Berl) 84(12):1047-54 PMID: 17024475
