Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli strain ER2566 bearing a deletion of the DHFR gene (folA) and thymidylate synthethase (thyA) as a His-tagged fusion protein | Streptococcus pneumoniae |
Protein Variants | Comment | Organism |
---|---|---|
H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T | Km or kcat (NADPH or dihydrofolate) much lower compared to wild-type, no difference in NADPH binding compared to wild-type, kcat of mutant is pH dependent (maximal rate at pH 7.0 similar to wild-type), inhibitor trimethoprim is bound tighter to mutant compared to wild-type | Streptococcus pneumoniae |
H33F | mutant shows a pH profile similar to DHFR from Escherichia coli, kcat values are pH independent over pH 6.0-7.5 | Streptococcus pneumoniae |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
trimethoprim | - |
Streptococcus pneumoniae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00063 | - |
dihydrofolate | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C, value below | Streptococcus pneumoniae | |
0.00075 | - |
NADPH | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C | Streptococcus pneumoniae | |
0.004 | - |
dihydrofolate | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae | |
0.0157 | - |
NADPH | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptococcus pneumoniae | Q54801 | - |
- |
Purification (Comment) | Organism |
---|---|
using Ni-NTA chromatography | Streptococcus pneumoniae |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
5,6,7,8-tetrahydrofolate + NADP+ = 7,8-dihydrofolate + NADPH + H+ | the product (tetrahydrofolate) dissociation step that is the rate-limiting step in Escherichia coli DHFR is significantly accelerated in spDHFR so that hydride transfer or a preceding step is rate-limiting | Streptococcus pneumoniae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
dihydrofolate + NADPH + H+ | - |
Streptococcus pneumoniae | tetrahydrofolate + NADP+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
dihydrofolate reductase | - |
Streptococcus pneumoniae |
spDHFR | - |
Streptococcus pneumoniae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Streptococcus pneumoniae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kcat for wild-type is pH-dependent, maximal kcat at pH 7.0 | Streptococcus pneumoniae | |
3.8 | - |
dihydrofolate | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C | Streptococcus pneumoniae | |
31.5 | - |
dihydrofolate | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at, maximal kcat at pH 7.0 for both wild-type and mutant | Streptococcus pneumoniae |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | - |
Streptococcus pneumoniae |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.000003 | - |
trimethoprim | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C | Streptococcus pneumoniae | |
0.000147 | - |
trimethoprim | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.00013 | - |
mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C | Streptococcus pneumoniae | trimethoprim | |
0.00048 | - |
wild-type, pH 7.0, 25°C | Streptococcus pneumoniae | trimethoprim |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2000 | - |
NADPH | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae | |
5000 | - |
NADPH | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C | Streptococcus pneumoniae | |
6000 | - |
dihydrofolate | mutant H26Y/Q60K/A77V/V78A/Q81H/Q91S/L100V/E133A/A149T, pH 7.0, 25°C, value above | Streptococcus pneumoniae | |
7200 | - |
dihydrofolate | wild-type, pH 7.0, 25°C | Streptococcus pneumoniae |