Literature summary for 1.5.1.3 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.

Application

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

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
N64F	site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview	Homo sapiens
N64S	site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview	Homo sapiens
Q35K	site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview	Homo sapiens
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
Q35S	site-directed mutagenesis, active site mutant showing enhanced activity compared to the wild-type enzyme, kinetic analysis, overview	Homo sapiens
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

Inhibitors	Comment	Organism
methotrexate	-	Homo sapiens
methotrexate	-	Pneumocystis carinii
methotrexate	-	Pneumocystis jirovecii
PY957	a trimethoprim analogue	Homo sapiens
PY957	a trimethoprim analogue	Pneumocystis carinii
PY957	a trimethoprim analogue	Pneumocystis jirovecii
trimethoprim	-	Homo sapiens
trimethoprim	-	Pneumocystis carinii
trimethoprim	-	Pneumocystis jirovecii

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P00374	-	-
Pneumocystis carinii	-	-	-
Pneumocystis jirovecii	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli by two steps of nickel affinity chromatography with Ulp1 protease cleavage after the first step	Pneumocystis carinii
recombinant His-tagged enzyme from Escherichia coli by two steps of nickel affinity chromatography with Ulp1 protease cleavage after the first step	Pneumocystis jirovecii
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)

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

Synonyms

Synonyms	Comment	Organism
DHFR	-	Pneumocystis carinii
DHFR	-	Pneumocystis jirovecii
DHFR	-	Homo sapiens

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
NADPH	-	Pneumocystis carinii
NADPH	-	Pneumocystis jirovecii
NADPH	-	Homo sapiens

Ki Value [mM]

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

kcat/KM [mM/s]

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