Cloned (Comment) | Organism |
---|---|
expression of the D120N and E28Q mutant plasmids in Escherichia coli strain AB 1909 | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
- |
Escherichia coli |
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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
purification of wild-type and histidine-tagged D120N and E28Q mutant enzymes | Escherichia coli |
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 | 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 Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | - |
Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | flavoprotein with FAD as cofactor | Escherichia coli | |
NADH | - |
Escherichia coli |