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Literature summary for 3.5.4.9 extracted from
- Residues involved in the mechanism of the bifunctional methylenetetrahydrofolate dehydrogenase-cyclohydrolase: The roles of glutamine 100 and aspartate 125 (2002), J. Biol. Chem., 277, 18703-18709.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of wild-type and mutant enzymes in Escherichia coli BL21 as C-terminally His-tagged enzymes | Homo sapiens |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D125A | site-directed mutagenesis, inactive mutant | Homo sapiens |
| D125C | site-directed mutagenesis, inactive mutant | Homo sapiens |
| D125E | site-directed mutagenesis, 95% reduced activity in the forward reaction compared to the wild-type enzyme | Homo sapiens |
| D125M | site-directed mutagenesis, inactive mutant | Homo sapiens |
| D125N | site-directed mutagenesis, inactive mutant | Homo sapiens |
| D125Q | site-directed mutagenesis, inactive mutant | Homo sapiens |
| K56Q/Q100K | site-directed mutagenesis, mutant shows no cyclohydrolase activity, but retains more than 2 thirds of wild-type dehydrogenase activity | Homo sapiens |
| additional information | construction of loop3G and loop5G deletion mutants which show 68.3% and 58.6% reduced activity, respectively, compared to the wild-type | Homo sapiens |
| Q100K | site-directed mutagenesis, inactive mutant | Homo sapiens |
| Q100M | site-directed mutagenesis, inactive mutant | Homo sapiens |
| Q100N | site-directed mutagenesis, inactive mutant | Homo sapiens |
| R250A | site-directed mutagenesis, 29.7% reduced activity in forward and reduced activity in the reverse reaction compared to the wild-type enzyme | Homo sapiens |
| Y240A | site-directed mutagenesis, 52,5% reduced activity in forward and reduced activity in the reverse reaction compared to the wild-type enzyme | Homo sapiens |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.05 | - |
5,10-methenyltetrahydrofolate | 30°C, recombinant mutant R250A | Homo sapiens |  |
| 0.06 | - |
5,10-methenyltetrahydrofolate | 30°C, recombinant wild-type enzyme | Homo sapiens | |
| 0.071 | - |
5,10-methenyltetrahydrofolate | 30°C, recombinant loop5G mutant | Homo sapiens | |
| 0.072 | - |
5,10-methenyltetrahydrofolate | 30°C, recombinant mutant Y240A | Homo sapiens | |
| 0.077 | - |
5,10-methenyltetrahydrofolate | 30°C, recombinant loop3G mutant | Homo sapiens | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Homo sapiens | P11586 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by Ni2+-chelate affinity chromatography | Homo sapiens |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| 5,10-methenyltetrahydrofolate + H2O = 10-formyltetrahydrofolate | catalytic mechanism, Glu100 is responsible for activation of the critical Lys56 for cyclohydrolase catalysis, Asp125 is involved in binding of the 10-formyltetrahydrofolate substrate | Homo sapiens |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| additional information | - |
channeling efficiency of wild-type and mutant enzymes | Homo sapiens |
| 51 | - |
recombinant loop3G mutant, cyclohydrolase forward reaction | Homo sapiens |
| 66.7 | - |
recombinant loop5G mutant, cyclohydrolase forward reaction | Homo sapiens |
| 76.5 | - |
recombinant mutant Y240A, cyclohydrolase forward reaction | Homo sapiens |
| 113 | - |
recombinant mutant R250A, cyclohydrolase forward reaction | Homo sapiens |
| 161 | - |
recombinant wild-type enzyme, cyclohydrolase forward reaction | Homo sapiens |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 5,10-methenyltetrahydrofolate + H2O | 5,10-methenyltetrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9, activity of the enzyme | Homo sapiens | 10-formyltetrahydrofolate | - |
r | |
| 5,10-methylenetetrahydrofolate + NADP+ | methylenetetrahydrofolate dehydrogenase (NADP+), EC 1.5.1.5, activity of the enzyme | Homo sapiens | 5,10-methenyltetrahydrofolate + NADPH + H+ | - |
r | |
| additional information | bifunctional enzyme, channeling efficiency | Homo sapiens | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| cyclohydrolase | - |
Homo sapiens |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Homo sapiens |
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