Cofactors

FAD.

Reaction Mechanism

methylenetetrahydrofolate reductase [NAD(P)H] By Reference

E. coli methylenetetrahydrofolate reductase (MTHFR) catalyses the NADH-dependent reduction of 5,10-methylenetetrahydrofolate (CH₂-H4folate) to 5 methyltetrahydrofolate (CH₃-H4folate) using the cofactor flavin adenine dinucleotide (FAD) as an intermediate hydride acceptor and donor. MTHFR is the only route of CH₃-H4folate which is used by methionine synthase to convert homocysteine to methionine.

E. coli MTHFR is a homotetramer that dissociates into dimers on dilution. Its catalytic domain is a (beta-alpha)8 barrel. Bacterial MTHFR enzymes are simpler than mammalian MTHFR in that the catatlytic domain constitutes the entire sequence, with no regulatory sequence present.




• Summary
• Step 1
• Step 2
• Step 3
• Products

E. coli MTHFR catalysis proceeds by a ping pong Bi-Bi reaction mechanism. The enzyme catalyses individual half-reactions, with the reduction of enzyme-bound FAD by NADH to form NAD⁺, which is released prior to CH₂-H4folate binding. CH₂-H4folate is thought to be converted to CH₃-H4folate by reduced E-FAD via a 5-iminium cation intermediate.

NADH binds at the si face of E-FAD(ox) cofactor. The 4S-hydrogen of NADH is transferred as a hydride to N5 of FAD, forming E-FAD(red) and NAD⁺. NAD⁺ dissociates from the enzyme. CH₂-H4folate also binds at the si face of FAD. N10 of CH₂-H4folate is thought to be protonated by Glu28, which leads to the opening of the five-membered imidazolidine ring of CH₂-H4folate to form the 5-iminium cation intermediate. The intermediate is stabilised by Asp120 and Glu28. A hydride is transferred from N5 of E-FAD(red) to the exocyclic methylene group (C11) of the stabilised 5-iminium cation intermediate to form the product CH₃-H4folate.

Catalytic Residues

AA	Uniprot	Uniprot Resid	PDB	PDB Resid
Ser	P0AEZ1	26	1zp3	26
Phe	P0AEZ1	223	1zp3	223
Glu	P0AEZ1	28	1zp3	28
Asp	P0AEZ1	120	1zp3	120
His	P0AEZ1	273	1zp3	273

Step Components

cofactor used, proton relay, intermediate formation, native state of cofactor regenerated, hydride transfer, decyclisation, aromatic bimolecular elimination, aromatic bimolecular nucleophilic addition, bimolecular nucleophilic addition, intermediate terminated, overall product formed, proton transfer, intramolecular elimination, native state of enzyme regenerated, overall reactant used, aromatic unimolecular elimination by the conjugate base



Step 1.

NAD(P) eliminates a hydride ion, which adds to FAD with concomitant deprotonation of water at the N1.



Step 2.

The substrate initiates the cleavage of the five-membered ring, forming the 5-iminium cation intermediate, with concomitant deprotonation of water through a proton relay chain of water, Glu28, His273 and Ser26.



Step 3.

Water deprotonates FAD, causing the elimination of a hydride which is added to the 5-iminium carbon of the intermediate.



Products.

The products of the reaction.

View Reaction Mechanisms in M-CSA

Reaction Parameters

• Kinetic Parameters

  Organism	KM Value [mM]	Substrate	Comment
  Homo sapiens	1146.5	NADPH	pH 6.6, 46°C, enzyme mutant HsMTHFR1-656

• Temperature

  Organism	Temperature Range	Comment
  Sus scrofa	25 - 45	methylenetetrahydrofolate reductase reaction, slow increase of activity between 25°C and 45°C

• pH

  Organism	pH Range	Comment
  Clostridium ljungdahlii	5 - 9	2% activity remain at pH 5.0, and 6% activity at pH 9.0
  Sus scrofa	5.8 - 8.6	about 60% of activity maximum at pH 5.8, about 50% of activity maximum at pH 8.6, menadione reductase activity
  Rattus norvegicus	6 - 8	pH 6: about 70% of activity maximum, pH 8: about 35% of activity maximum with 5-methyl-5,6,7,8-tetrahydropteroylmonoglutamate, about 65% of activity maximum with 5-methyl-5,6,7,8-tetrahydropteroylpentaglutamate

Associated Proteins

Citations

• Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase.
• 5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity.
• Nicotinamide riboside supplementation is not associated with altered methylation homeostasis in Parkinson's disease.
• ALDH2 rs671 and MTHFR rs1801133 polymorphisms are risk factors for arteriosclerosis in multiple arteries.
• One-carbon metabolizing enzyme ALDH1L1 influences mitochondrial metabolism through 5-aminoimidazole-4-carboxamide ribonucleotide accumulation and serine depletion, contributing to tumor suppression.
• A Third Way of Energy Conservation in Acetogenic Bacteria.
• Microbial detoxification of lignocellulosic biomass hydrolysates: Biochemical and molecular aspects, challenges, exploits and future perspectives.
• Targeting serine-glycine-one-carbon metabolism as a vulnerability in cancers.
• Serine Metabolic Reprogramming in Tumorigenesis, Tumor Immunity, and Clinical Treatment
• Anaerobic Biohydrogenation of Isoprene by Acetobacterium wieringae Strain Y.
• Cloneable inorganic nanoparticles.