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Literature summary for 1.5.1.20 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)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
-
Escherichia coli

Protein Variants

Protein Variants Comment Organism
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
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]

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

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
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+
-
?
5,10-methylenetetrahydrofolate + reduced acceptor reverse reaction: menadione as electron acceptor Escherichia coli 5-methyltetrahydrofolate + oxidized acceptor
-
r
5,10-methylenetetrahydrofolate + reduced acceptor forward reaction: NADH as reduced acceptor Escherichia coli 5-methyltetrahydrofolate + oxidized acceptor
-
r
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]

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

Cofactor

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