Literature summary for 1.5.1.3 extracted from

Frey, K.M.; Liu, J.; Lombardo, M.N.; Bolstad, D.B.; Wright, D.L.; Anderson, A.C.
Crystal structures of wild-type and mutant methicillin-resistant Staphylococcus aureus dihydrofolate reductase reveal an alternate conformation of NADPH that may be linked to trimethoprim resistance (2009), J. Mol. Biol., 387, 1298-1308.

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of His-tagged wild-type and mutant enzmyes in Escherichia coli strain BL21(DE3) as soluble proteins	Staphylococcus aureus

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and mutant enzymes bound to cofactor NADPH and inhibitors 5, 8, 10, and 15, hanging-drop vaporization method, 12 mg/ml protein is incubated with 1 mM inhibitor and 2 mM NADPH on ice for 2 h, mixing of equal volumes of the protein:ligand:NADPH solution with an optimized crystallization solution consisting of 15% PEG 10000, 150 mM sodium acetate, 100 mM MES, pH 6.5, and 5% butyrlactone, 5-7 days X-ray diffraction structure determination and analysis at 1.69-2.35 A resolutions, overview	Staphylococcus aureus

Crystallization (Comment)

Organism

purified recombinant wild-type and mutant enzymes bound to cofactor NADPH and inhibitors 5, 8, 10, and 15, hanging-drop vaporization method, 12 mg/ml protein is incubated with 1 mM inhibitor and 2 mM NADPH on ice for 2 h, mixing of equal volumes of the protein:ligand:NADPH solution with an optimized crystallization solution consisting of 15% PEG 10000, 150 mM sodium acetate, 100 mM MES, pH 6.5, and 5% butyrlactone, 5-7 days X-ray diffraction structure determination and analysis at 1.69-2.35 A resolutions, overview

Staphylococcus aureus

Inhibitors

Inhibitors	Comment	Organism
3-(2,4-diamino-6-methylpyrimidin-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol	-	Staphylococcus aureus
5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-ethylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-propylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]pyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2-methoxy-2',6'-dimethylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2-methoxy-2'-methylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(2-methoxybiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(5-methoxy-2',6'-dimethylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(5-methoxy-2'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(5-methoxy-4'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-(5-methoxybiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
5-[3-methoxy-3-(3,4,5-trimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine	-	Staphylococcus aureus
6-methyl-5-[3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine	-	Staphylococcus aureus
6-methyl-5-[3-(3,4,5-trimethoxyphenyl)pent-1-yn-1-yl]pyrimidine-2,4-diamine	-	Staphylococcus aureus
6-methyl-5-[3-methyl-3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine	-	Staphylococcus aureus
additional information	structure based inhibitor development of propargyl derivatives of trimethoprim for inhibition of resistant enzyme, overview	Staphylococcus aureus
trimethoprim	inhibition of the wild-type enzyme, but 74fold less of the resistant mutant F98Y	Staphylococcus aureus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
7,8-dihydrofolate + NADPH + H+	Staphylococcus aureus	-	5,6,7,8-tetrahydrofolate + NADP+	-	?

Organism

Organism	UniProt	Comment	Textmining
Staphylococcus aureus	P0A016	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzmyes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Staphylococcus aureus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
7,8-dihydrofolate + NADPH + H+	-	Staphylococcus aureus	5,6,7,8-tetrahydrofolate + NADP+	-	?

Synonyms

Synonyms	Comment	Organism
DHFR	-	Staphylococcus aureus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Staphylococcus aureus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Staphylococcus aureus

Cofactor

Cofactor	Comment	Organism	Structure
NADPH	binding structure and comformation of wild-type and mutant enzyme	Staphylococcus aureus

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.000023	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	trimethoprim
0.000042	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(5-methoxybiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.000048	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-propylpyrimidine-2,4-diamine
0.000061	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(5-methoxybiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.000064	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	6-methyl-5-[3-methyl-3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine
0.000068	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-ethylpyrimidine-2,4-diamine
0.000073	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(5-methoxy-2'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.000073	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(5-methoxy-2',6'-dimethylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00012	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	6-methyl-5-[3-(3,4,5-trimethoxyphenyl)pent-1-yn-1-yl]pyrimidine-2,4-diamine
0.00017	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(5-methoxybiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00017	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	6-methyl-5-[3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine
0.00017	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2-methoxybiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00018	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(5-methoxy-2',6'-dimethylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00019	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(5-methoxybiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00019	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(5-methoxy-2'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00027	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00027	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.00041	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(5-methoxy-4'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00041	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2-methoxy-2',6'-dimethylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.0005	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-(2-methoxy-2'-methylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00055	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-propylpyrimidine-2,4-diamine
0.00067	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-ethylpyrimidine-2,4-diamine
0.00073	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	5-[3-methoxy-3-(3,4,5-trimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00075	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	6-methyl-5-[3-methyl-3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine
0.00077	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2-methoxybiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00096	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(5-methoxy-4'-methylbiphenyl-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.0011	-	pH 7.0, 25°C, wild-type enzyme	Staphylococcus aureus	3-(2,4-diamino-6-methylpyrimidin-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol
0.0012	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	6-methyl-5-[3-(3,4,5-trimethoxyphenyl)but-1-yn-1-yl]pyrimidine-2,4-diamine
0.0013	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.00142	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2-methoxy-2'-methylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.0017	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	trimethoprim
0.0039	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	6-methyl-5-[3-(3,4,5-trimethoxyphenyl)pent-1-yn-1-yl]pyrimidine-2,4-diamine
0.0041	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	3-(2,4-diamino-6-methylpyrimidin-5-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-ol
0.00613	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2-methoxy-2',6'-dimethylbiphenyl-4-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
0.0069	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-(2,5-dimethoxyphenyl)prop-1-yn-1-yl]pyrimidine-2,4-diamine
0.012	-	pH 7.0, 25°C, mutant F98Y	Staphylococcus aureus	5-[3-methoxy-3-(3,4,5-trimethoxyphenyl)prop-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine