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Literature summary for 1.5.1.3 extracted from
- High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase (1999), Biochim. Biophys. Acta, 1429, 401-410.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| high expression rate of wild-type and mutants from expression vector pCOCK | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| L16M | site-directed mutagenesis, same kinetic properties like the wild-type enzyme | Escherichia coli |
| L16M/L20M | site-directed mutagenesis, double mutant, elevated turnover number and specific activity | Escherichia coli |
| L16M/L20M/L42M | site-directed mutagenesis, triple mutant, elevated turnover number and specific activity | Escherichia coli |
| L16SeM | site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme | Escherichia coli |
| L16SeM/L20SeM | site-directed mutagenesis, double mutant, with selenomethionine, elevated turnover number and specific activity | Escherichia coli |
| L16SeM/L20SeM/L42SeM | site-directed mutagenesis, triple mutant, with selenomethionine, elevated turnover number and specific activity | Escherichia coli |
| L20M | site-directed mutagenesis, elevated turnover number and specific activity | Escherichia coli |
| L20SeM | site-directed mutagenesis, with selenomethionine, elevated turnover number and specific activity | Escherichia coli |
| L42M | site-directed mutagenesis, same kinetic properties like the wild-type enzyme | Escherichia coli |
| L42SeM | site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme | Escherichia coli |
| L92M | site-directed mutagenesis, same kinetic properties like the wild-type enzyme | Escherichia coli |
| L92SeM | site-directed mutagenesis, with selenomethionine, same kinetic properties like the wild-type enzyme | Escherichia coli |
General Stability
| General Stability | Organism |
|---|---|
| NADPH stabilizes | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | selenomethionine-containing mutants | Escherichia coli | |
| 0.00097 | - |
7,8-dihydrofolate | wild-type enzyme from plasmid | Escherichia coli |  |
| 0.00123 | - |
7,8-dihydrofolate | mutant L92M from plasmid | Escherichia coli | |
| 0.00167 | - |
7,8-dihydrofolate | double mutant L16M/L20M from plasmid | Escherichia coli | |
| 0.00171 | - |
7,8-dihydrofolate | mutant L42M from plasmid | Escherichia coli | |
| 0.00222 | - |
7,8-dihydrofolate | mutant L16M from plasmid | Escherichia coli | |
| 0.00228 | - |
NADPH | double mutant L16M/L20M from plasmid | Escherichia coli | |
| 0.00232 | - |
NADPH | mutant L16M from plasmid | Escherichia coli | |
| 0.00252 | - |
NADPH | wild-type enzyme from plasmid | Escherichia coli | |
| 0.0027 | - |
NADPH | mutant L92M from plasmid | Escherichia coli | |
| 0.00289 | - |
7,8-dihydrofolate | mutant L20M from plasmid | Escherichia coli | |
| 0.003 | - |
NADPH | mutant L42M from plasmid | Escherichia coli | |
| 0.00369 | - |
7,8-dihydrofolate | triple mutant L16M/L20M/L42M from plasmid | Escherichia coli | |
| 0.0043 | - |
NADPH | mutant L20M from plasmid | Escherichia coli | |
| 0.00456 | - |
NADPH | triple mutant L16M/L20M/L42M from plasmid | Escherichia coli | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 18000 | - |
gel filtration | Escherichia coli |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
wild-type and mutants | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| wild-type and mutants from high expression vector pCOCK, large scale purification | Escherichia coli |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| additional information | - |
selenomethionine-containing mutants | Escherichia coli |
| 45.9 | - |
wild-type enzyme from plasmid | Escherichia coli |
| 90.2 | - |
double mutant L16M/L20M from plasmid | Escherichia coli |
| 92.7 | - |
mutant L20M from plasmid | Escherichia coli |
| 172 | - |
triple mutant L16M/L20M/L42M from plasmid | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 7,8-dihydrofolate + NADPH | - |
Escherichia coli | 5,6,7,8-tetrahydrofolate + NADP+ | - |
? | |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
with NADPH bound all mutants retain activity longer than the wild-type enzyme, half-life of the wild-type enzyme 32 days | Escherichia coli |
| 37 | - |
without NADPH, 50% loss of activity of wild-type and mutant enzymes after 60 h | Escherichia coli |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | selenomethionine-containing mutants | Escherichia coli | |
| 13.7 | - |
7,8-dihydrofolate | wild-type enzyme from plasmid | Escherichia coli | |
| 24.6 | - |
7,8-dihydrofolate | mutant L20M from plasmid | Escherichia coli | |
| 25.2 | - |
7,8-dihydrofolate | double mutant L16M/L20M from plasmid | Escherichia coli | |
| 52.7 | - |
7,8-dihydrofolate | triple mutant L16M/L20M/L42M from plasmid | Escherichia coli | |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| NADPH | wild-type and mutant enzymes | Escherichia coli | |
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