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Literature summary extracted from

  • Zuo, C.; Jolly, A.L.; Nikolova, A.P.; Satzer, D.I.; Cao, S.; Sanchez, J.S.; Ballou, D.P.; Trimmer, E.E.
    A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli (2018), Arch. Biochem. Biophys., 642, 63-74 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.15 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain AB1909 Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.54 structures of the enzyme complexes of wild-type with NADH and mutant E28Q with methytetrahydrofolate. Residue Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective halfreactions Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.15 Q183A site-directed mutagenesis, the Gln183Ala variant exhibits a 6-10fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity compared to wild-type enzyme. The oxidative half-reaction of the Gln183Ala mutant is considered reversible, and the enzyme can catalyze either of two half reactions involving folate: reduction of CH2-H4folate as part of the physiological oxidoreductase reaction or oxidation of CH3-H4folate as part of the CH3-H4folate-menadione oxidoreductase reaction, comparisons of half-reaction kinetics of wild-type and mutant enzymes Escherichia coli
1.5.1.15 Q183E site-directed mutagenesis, comparisons of half-reaction kinetics of wild-type and mutant enzymes, the Gln183Glu mutant displays catalytic constants within 3fold of the wild-type enzyme enzyme Escherichia coli
1.5.1.54 Q183A in the reductive half-reaction, NADH binding affinity and the rate of flavin reduction are not hindered by the mutation. Q183A exhibits a 6-10fold lower rate of folate reduction and binds methylentetrahydrofolate with 7-fold lower affinity Escherichia coli
1.5.1.54 Q183E in the reductive half-reaction, NADH binding affinity and the rate of flavin reduction are not hindered by the mutation and Gln183 plays a minor in the oxidative half-reaction. The mutant displays catalytic constants within 3fold of the wild-type enzyme Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.15 5,10-methylenetetrahydrofolate substrate inhibition of wild-type enzyme and mutant Q183E, no inhibition of mutant Q183A Escherichia coli
1.5.1.15 menadione substrate inhibition of wild-type enzyme and mutant Q183A, no inhibition of mutant Q183E Escherichia coli
1.5.1.15 NADH substrate inhibition Escherichia coli
1.5.1.54 5,10-methylenetetrahydrofolate substrate inhibition Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.15 additional information
-
 additional information Michaelis-Menten steady-state kinetic analysis, comparisons of half-reaction kinetics of wild-type and mutant enzymes Escherichia coli
1.5.1.15 0.0004
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.0035
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.0066
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.01
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.0151
-
 NADH pH 7.2, 25°C, recombinant mutant Q183A, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.0204
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.025
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.03
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.108
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183E, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.115
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183E, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.29
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183A, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.29
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.54 0.0004
-
 5,10-methylenetetrahydrofolate wild-type, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.0035
-
 NADH wild-type, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.0066
-
 NADH mutant Q183E, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.01
-
 NADH mutant Q183A, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.104
-
 5,10-methylenetetrahydrofolate mutant Q183A, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.108
-
 5,10-methylenetetrahydrofolate mutant Q183E, pH 7.2, 4°C Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.15 5,10-methylenetetrahydrofolate + NAD+ Escherichia coli
-
 5,10-methenyltetrahydrofolate + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.15 Escherichia coli
-
-
-
1.5.1.54 Escherichia coli P0AEZ1 cf. EC 1.5.1.20
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.15 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain AB1909 Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
1.5.1.15 5,10-methylenetetrahydrofolate + NAD+ = 5,10-methenyltetrahydrofolate + NADH + H+ the reaction catalyzed by Escherichia coli MTHFR can be divided into a reductive half-reaction and an oxidative half-reaction, respectively, in which the FAD cofactor acts as an intermediate electron acceptor and donor. The enzyme employs a ping-pong bi-bi kinetic mechanism, in which the release of NAD+, the product of the first half-reaction, precedes binding of the substrate CH2-H4folate in the second half-reaction. Rapid-reaction kinetic studies have established that the enzyme catalyzes these half-reactions at rates consistent with the observed rate of overall turnover. Proposed mechanism for the oxidative half-reaction, overview Escherichia coli
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.15 5,10-methylenetetrahydrofolate + NAD+
-
 Escherichia coli 5,10-methenyltetrahydrofolate + NADH + H+
-
 r
1.5.1.15 5,10-methylenetetrahydrofolate + NAD+ enzyme binding structure analysis, overview Escherichia coli 5,10-methenyltetrahydrofolate + NADH + H+
-
 r
1.5.1.15 additional information the reaction catalyzed by Escherichia coli MTHFR can be divided into a reductive half-reaction and an oxidative half-reaction, respectively, in which the FAD cofactor acts as an intermediate electron acceptor and donor. For enzyme assay, menadione is used as an alternate electron acceptor, MTHFR oxidizes NADH to NAD+, and the reaction can be monitored at 343 nm by the decrease in NADH absorbance Escherichia coli ?
-
-
1.5.1.54 5,10-methylenetetrahydrofolate + NADH + H+
-
 Escherichia coli 5-methyltetrahydrofolate + NAD+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.5.1.15 homotetramer 4 * 33000, beta8alpha8 barrel, SDS-PAGE Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
1.5.1.15 methylenetetrahydrofolate reductase
-
 Escherichia coli
1.5.1.15 MTHFR
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.15 25
-
 assay at Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.15 0.15
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.29
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183A, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.29
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.47
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 2.2
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 2.2
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 2.7
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 2.7
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183E, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 14
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 15.3
-
 NADH pH 7.2, 25°C, recombinant mutant Q183A, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 18.2
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.54 0.29
-
 NADH mutant Q183A, pH 7.2, 4°C Escherichia coli
1.5.1.54 2.2
-
 NADH wild-type, pH 7.2, 4°C Escherichia coli
1.5.1.54 2.7
-
 NADH mutant Q183E, pH 7.2, 4°C Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.15 7.2
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.15 FAD a flavoprotein, the FAD cofactor is bound in the center of the barrel with only the si face of the isoalloxazine ring exposed to solvent Escherichia coli
1.5.1.15 additional information preference for NADH over NADPH Escherichia coli
1.5.1.15 NAD+
-
 Escherichia coli
1.5.1.15 NADH enzyme binding structure analysis, overview Escherichia coli

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.5.1.15 0.0017
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.0032
-
 NADH pH 7.2, 25°C, recombinant mutant Q183A, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.023
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.054
-
 menadione pH 7.2, 25°C, recombinant wild-type, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.061
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.15 0.081
-
 menadione pH 7.2, 25°C, recombinant mutant Q183A, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 0.094
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183E, NADH-CH2-H4folate oxidoreductase assay Escherichia coli
1.5.1.54 0.061
-
 5,10-methylenetetrahydrofolate wild-type, pH 7.2, 4°C Escherichia coli
1.5.1.54 0.094
-
 5,10-methylenetetrahydrofolate mutant Q183E, pH 7.2, 4°C Escherichia coli

General Information

EC Number General Information Comment Organism
1.5.1.15 evolution the Escherichia coli enzyme differs from the characterized pig, human, and yeast MTHFRs (EC 1.5.1.5) in its preference for NADH over NADPH as substrate and by the absence of a regulatory domain. Structures of Ered(wild-type)-NADH and Eox(Glu28Gln)-CH3-H4folate complexes reveal that the ligands occupy partially overlapping sites at the si face of the FAD. Thus, the binding of one substrate to the enzyme prevents the binding of the other substrate, consistent with the ping-pong bi-bi kinetic mechanism. But the structures reveal that the active site conformations of NADH and CH3-H4folate are remarkably different Escherichia coli
1.5.1.15 additional information a role for residue glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli. Residue Gln183 may participate in NADH binding, based on the hydrogen bonding interactions between the Gln side chain and the carboxamide of NADH observed in the X-ray structure Escherichia coli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.15 1.3
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant mutant Q183A, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 15.67
-
 5,10-methylenetetrahydrofolate pH 7.2, 25°C, recombinant wild-type, CH3-H4folate-menadione oxidoreductase assay Escherichia coli
1.5.1.15 686.3
-
 NADH pH 7.2, 25°C, recombinant mutant Q183A, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 728
-
 NADH pH 7.2, 25°C, recombinant wild-type, NADH-menadione oxidoreductase assay Escherichia coli
1.5.1.15 1013.3
-
 NADH pH 7.2, 25°C, recombinant mutant Q183E, NADH-menadione oxidoreductase assay Escherichia coli