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Literature summary extracted from

  • Cody, V.; Pace, J.; Makin, J.; Piraino, J.; Queener, S.F.; Rosowsky, A.
    Correlations of inhibitor kinetics for Pneumocystis jirovecii and human dihydrofolate reductase with structural data for human active site mutant enzyme complexes (dagger) (double dagger) (2009), Biochemistry, 48, 1702-1711.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.5.1.3 drug development the essential enzyme is a target for development of specific inhibitors for treatment of the causative agent in AIDS pneumonia Pneumocystis carinii
1.5.1.3 drug development the essential enzyme is a target for development of specific inhibitors for treatment of the causative agent in AIDS pneumonia Pneumocystis jirovecii

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 expression of His-tagged enzyme in Escherichia coli Pneumocystis carinii
1.5.1.3 expression of His-tagged enzyme in Escherichia coli Pneumocystis jirovecii
1.5.1.3 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.3 purified recombinant wild-type and mutant enzymes Q35K, Q35S/N64F, and Q35S/N64S in complex with NADPH and inhibitor PY957, hanging drop vapor diffusion method using, 0.010 ml protein solution containing 4.2 mg/ml for the single mutant protein and 6.6 mg/ml for the double mutant proteins in 100 mM K2HPO4, pH 6.9, with 30% saturated ammonium sulfate with a 10:1 molar excess of NADPH and PY957, are mixed with reservoir solution containing 100 mM K2HPO4, pH 6.9, and 60% saturated ammonium sulfate with 3% v/v ethanol, 3 days at 14°C, X-ray diffraction structure determination and analysis at 1.2-2.0 A resolution Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.3 N64F site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview Homo sapiens
1.5.1.3 N64S site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview Homo sapiens
1.5.1.3 Q35K site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview Homo sapiens
1.5.1.3 Q35K/N64F site-directed mutagenesis, active site mutant analogues to the enzyme from Pneumocystis carinii, shows enhanced activity compared to the wild-type enzymekinetic analysis, overview Homo sapiens
1.5.1.3 Q35S site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview Homo sapiens
1.5.1.3 Q35S/N64S site-directed mutagenesis, active site mutant analogues to the enzyme from Pneumocystis jirovecii, shows enhanced activity compared to the wild-type enzyme, kinetic analysis, overview Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 methotrexate
-
 Homo sapiens
1.5.1.3 methotrexate
-
 Pneumocystis carinii
1.5.1.3 methotrexate
-
 Pneumocystis jirovecii
1.5.1.3 PY957 a trimethoprim analogue Homo sapiens
1.5.1.3 PY957 a trimethoprim analogue Pneumocystis carinii
1.5.1.3 PY957 a trimethoprim analogue Pneumocystis jirovecii
1.5.1.3 trimethoprim
-
 Homo sapiens
1.5.1.3 trimethoprim
-
 Pneumocystis carinii
1.5.1.3 trimethoprim
-
 Pneumocystis jirovecii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.3 additional information
-
 additional information steady-state kinetics of wild-type and mutant enzymes Homo sapiens
1.5.1.3 0.00018
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 0.00049
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 0.00076
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 0.00081
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 0.0011
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 0.0016
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 0.0027
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Homo sapiens
1.5.1.3 0.0028
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 0.004
-
 NADPH pH 7.4, 37°C, wild-type enzyme Homo sapiens
1.5.1.3 0.0044
-
 NADPH pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 0.0046
-
 NADPH pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 0.0049
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 0.0052
-
 NADPH pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 0.0055
-
 NADPH pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 0.0063
-
 NADPH pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 0.0085
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 0.0092
-
 NADPH pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 0.013
-
 NADPH pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 0.0145
-
 NADPH pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 0.0229
-
 NADPH pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Pneumocystis carinii
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 ?
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Pneumocystis jirovecii
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 ?
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Homo sapiens
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Homo sapiens P00374
-
-
1.5.1.3 Pneumocystis carinii
-
-
-
1.5.1.3 Pneumocystis jirovecii
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 recombinant His-tagged enzyme from Escherichia coli by two steps of nickel affinity chromatography with Ulp1 protease cleavage after the first step Pneumocystis carinii
1.5.1.3 recombinant His-tagged enzyme from Escherichia coli by two steps of nickel affinity chromatography with Ulp1 protease cleavage after the first step Pneumocystis jirovecii
1.5.1.3 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, methotrexate affinity chromatography, dialysis and anion exchange chromatography Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Pneumocystis carinii 5,6,7,8-tetrahydrofolate + NADP+
-
 ?
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Pneumocystis jirovecii 5,6,7,8-tetrahydrofolate + NADP+
-
 ?
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Homo sapiens 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.5.1.3 DHFR
-
 Pneumocystis carinii
1.5.1.3 DHFR
-
 Pneumocystis jirovecii
1.5.1.3 DHFR
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.3 37
-
 assay at Pneumocystis carinii
1.5.1.3 37
-
 assay at Pneumocystis jirovecii
1.5.1.3 37
-
 assay at Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.3 3 6 7,8-dihydrofolate pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 27
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 35
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 40
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme and mutant N64F Homo sapiens
1.5.1.3 43
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 47
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 48
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 53
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 284
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.3 7.4
-
 assay at Pneumocystis carinii
1.5.1.3 7.4
-
 assay at Pneumocystis jirovecii
1.5.1.3 7.4
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADPH
-
 Pneumocystis carinii
1.5.1.3 NADPH
-
 Pneumocystis jirovecii
1.5.1.3 NADPH
-
 Homo sapiens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.5.1.3 additional information
-
 additional information comparison of inhibition kinetics of wild-type and mutant enzymes with those of the enzyme from Homo sapiens Pneumocystis jirovecii
1.5.1.3 additional information
-
 additional information comparison of inhibition kinetics of wild-type and mutant enzymes with those of the enzymes Pneumocystis carinii and Pneumocystis jirovecii Homo sapiens
1.5.1.3 additional information
-
 additional information comparison of inhibition kinetics of wild-type enzyme with those of wild-type and mutant enzyme from Homo sapiens Pneumocystis carinii
1.5.1.3 0.000000002
-
 methotrexate pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 0.000000003
-
 methotrexate pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 0.000000014
-
 methotrexate pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 0.000000019
-
 methotrexate pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 0.000000029
-
 methotrexate pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 0.000000042
-
 methotrexate pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 0.000000047
-
 methotrexate pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 0.000000047
-
 methotrexate pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 0.000000063
-
 methotrexate pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 0.000000093
-
 methotrexate pH 7.4, 37°C, wild-type enzyme Homo sapiens
1.5.1.3 0.0000052
-
 PY957 pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 0.0000076
-
 PY957 pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 0.0000083
-
 PY957 pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 0.000013
-
 PY957 pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 0.0000219
-
 PY957 pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 0.000023
-
 PY957 pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 0.0000263
-
 PY957 pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 0.000043
-
 trimethoprim pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 0.000066
-
 PY957 pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 0.000075
-
 trimethoprim pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 0.00023
-
 PY957 pH 7.4, 37°C, wild-type enzyme Homo sapiens
1.5.1.3 0.00027
-
 trimethoprim pH 7.4, 37°C, mutant N64S Homo sapiens
1.5.1.3 0.00035
-
 PY957 pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 0.00039
-
 trimethoprim pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 0.00049
-
 trimethoprim pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 0.00065
-
 trimethoprim pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 0.0008
-
 trimethoprim pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 0.0012
-
 trimethoprim pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 0.0018
-
 trimethoprim pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 0.0052
-
 trimethoprim pH 7.4, 37°C, wild-type enzyme Homo sapiens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.3 4000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant K37Q Pneumocystis carinii
1.5.1.3 15000
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Homo sapiens
1.5.1.3 17000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S/N64S Homo sapiens
1.5.1.3 19000
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis jirovecii
1.5.1.3 33000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35S Homo sapiens
1.5.1.3 49000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant N64F Homo sapiens
1.5.1.3 58000
-
 7,8-dihydrofolate pH 7.4, 37°C, wild-type enzyme Pneumocystis carinii
1.5.1.3 63000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K Homo sapiens
1.5.1.3 88000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant Q35K/N64F Homo sapiens
1.5.1.3 261000
-
 7,8-dihydrofolate pH 7.4, 37°C, mutant N64S Homo sapiens