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Literature summary extracted from

  • Trimmer, E.E.; Ballou, D.P.; Ludwig, M.L.; Matthews, R.G.
    Folate activation and catalysis in methylenetetrahydrofolate reductase from Escherichia coli: Roles for aspartate 120 and glutamate 28 (2001), Biochemistry, 40, 6216-6226.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.20 expression of the D120N and E28Q mutant plasmids in Escherichia coli strain AB 1909 Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.20
-
 Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.20 D120N mutant with 150fold decreased activity in the physiological NADH-CH2-H4folate oxidoreductase reaction, enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, it binds methylenetetrahydrofolate in its ring-closed form, but no conversion to the 5-iminium cation, enzyme-bound FAD is more easily reduced and more difficult reoxidized than FAD of wild-type enzyme Escherichia coli
1.5.1.20 E28Q mutant enzyme is unable to catalyze reduction of methylenetetrahydrofolate and is inactive in the physiological NADH-CH2-H4folate oxidoreductase reaction, it binds methyltetrahydrofolate, but reduces not the FAD cofactor, 240fold decrease in NADH-menadione oxidoreductase activity Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.20 0.0005
-
 5,10-methylenetetrahydrofolate wild-type enzyme Escherichia coli
1.5.1.20 0.005
-
 NADH D120N mutant enzyme Escherichia coli
1.5.1.20 0.02
-
 NADH wild-type enzyme Escherichia coli
1.5.1.20 0.027
-
 5,10-methylenetetrahydrofolate D120N mutant enzyme Escherichia coli
1.5.1.20 0.085
-
 5-methyltetrahydrofolate wild-type enzyme Escherichia coli
1.5.1.20 0.16
-
 5-methyltetrahydrofolate D120N mutant enzyme Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.20 5,10-methylenetetrahydrofolate + NADH Escherichia coli physiological direction is the 5-methyltetrahydrofolate formation by transfer of reducing equivalents from NADH to the enzyme-bound FAD and from reduced FAD to methylenetetrahydrofolate 5-methyltetrahydrofolate + NAD+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.20 Escherichia coli
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.20 purification of wild-type and histidine-tagged D120N and E28Q mutant enzymes Escherichia coli

Reaction

EC Number Reaction Comment Organism Reaction ID
1.5.1.20 5-methyltetrahydrofolate + NAD(P)+ = 5,10-methylenetetrahydrofolate + NAD(P)H + H+ ping-pong bi-bi mechanism, in which NAD(P)+ release precedes the binding of methylenetetrahydrofolate Escherichia coli
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.20 5,10-methylenetetrahydrofolate + NADH physiological direction is the 5-methyltetrahydrofolate formation by transfer of reducing equivalents from NADH to the enzyme-bound FAD and from reduced FAD to methylenetetrahydrofolate Escherichia coli 5-methyltetrahydrofolate + NAD+
-
 ?
1.5.1.20 5,10-methylenetetrahydrofolate + reduced acceptor reverse reaction: menadione as electron acceptor Escherichia coli 5-methyltetrahydrofolate + oxidized acceptor
-
 r
1.5.1.20 5,10-methylenetetrahydrofolate + reduced acceptor forward reaction: NADH as reduced acceptor Escherichia coli 5-methyltetrahydrofolate + oxidized acceptor
-
 r
1.5.1.20 additional information catalytic mechanism, Asp-120 and Glu-28 at the flavin active site are relevant to catalysis, Asp-120: located near the enzyme-bound FAD, role in catalysis of folate reduction and in stabilization of the folate intermediate 5-iminium cation, Glu-28: located near N10 of the folate, general acid catalyst to aid in 5-iminium cation formation Escherichia coli ?
-
 ?

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.20 additional information
-
 additional information
-
 Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.20 FAD flavoprotein with FAD as cofactor Escherichia coli
1.5.1.20 NADH
-
 Escherichia coli