Enzyme - Methylenetetrahydrofolate reductase (NAD(P)H)

Alternative Name(s)
  • 5-methyltetrahydrofolate:NAD(+) oxidoreductase.
  • Methylenetetrahydrofolate reductase.
  • phosphate) reductase.
  • Methylenetetrahydrofolate (reduced nicotinamide adenine dinucleotide
  • 5-methyltetrahydrofolate:NADP(+) oxidoreductase.
  • 5,10-CH(2)-H(4)folate reductase.
  • MTHFR.
  • 5-methyltetrahydrofolate:(acceptor) oxidoreductase.
  • Methylenetetrahydrofolate (reduced riboflavin adenine dinucleotide)
  • Methylenetetrahydrofolate reductase (NADPH).
  • 5,10-methylenetetrahydrofolate reductase (FADH(2)).
  • reductase.
  • 5,10-methylenetetrahydrofolate reductase.
  • 5,10-methylenetetrahydrofolate reductase (NADPH).
  • N(5,10)-methylenetetrahydrofolate reductase.
  • 5-methyltetrahydrofolate:NAD oxidoreductase.
  • Methylenetetrahydrofolic acid reductase.
  • Methylenetetrahydrofolate reductase (NADPH(2)).
  • 5,10-methylenetetrahydropteroylglutamate reductase.
  • N(5),N(10)-methylenetetrahydrofolate reductase.
  • 5,10-methylenetetrahydrofolic acid reductase.

Catalytic Activity

(6S)-5-methyl-5,6,7,8-tetrahydrofolate + NAD(+) = (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate + H(+) + NADH



Reaction Mechanism

    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.

    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.


    The products of the reaction.

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

Protein name Organism
Probable methylenetetrahydrofolate reductase Caenorhabditis elegans
5,10-methylenetetrahydrofolate reductase Escherichia coli (strain K12)
Methylenetetrahydrofolate reductase 1 Mouse-ear cress
Methylenetetrahydrofolate reductase 2 Mouse-ear cress
Methylenetetrahydrofolate reductase Human