Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.5.1.3 extracted from

  • Lee, J.; Yennawar, N.H.; Gam, J.; Benkovic, S.J.
    Kinetic and structural characterization of dihydrofolate reductase from Streptococcus pneumoniae (2010), Biochemistry, 49, 195-206.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

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

Inhibitors Comment Organism Structure
trimethoprim
-
Streptococcus pneumoniae

KM Value [mM]

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

Organism UniProt Comment Textmining
Streptococcus pneumoniae Q54801
-
-

Purification (Commentary)

Purification (Comment) Organism
using Ni-NTA chromatography Streptococcus pneumoniae

Reaction

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 and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
dihydrofolate + NADPH + H+
-
Streptococcus pneumoniae tetrahydrofolate + NADP+
-
?

Synonyms

Synonyms Comment Organism
dihydrofolate reductase
-
Streptococcus pneumoniae
spDHFR
-
Streptococcus pneumoniae

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Streptococcus pneumoniae

Turnover Number [1/s]

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

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

Cofactor Comment Organism Structure
NADPH
-
Streptococcus pneumoniae

Ki Value [mM]

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 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 [mM/s]

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