Literature summary for 1.5.1.3 extracted from

Bourne, C.; Wakeham, N.; Webb, N.; Nammalwar, B.; Bunce, R.; Berlin, K.; Barrow, W.
The structure and competitive substrate inhibition of dihydrofolate reductase from Enterococcus faecalis reveal restrictions to cofactor docking (2014), Biochemistry, 53, 1228-1238 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of C-terminally StrepII-tagged DHFR in Escherichia coli strain BL21(DE3)pLysS	Enterococcus faecalis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged enzyme in apoform or complex with inhibitor RAB, sitting drop vapor diffusion method, for the apoenzyme, mixing of 800 nl of 16.5 mg/ml protein in 20 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM DTT, with 800 nl of well solution containing 5% PEG 3350 and 1.5 M ammonium citrate dibasic, pH 7.0, 1 week at room temperature, crystals complexed with RAB are grown from 1.1 M ammonium tartrate dibasic, pH 7.0, at 4°C in 2-3 months, X-ray diffraction structure determination and analysis at 2.1-2.3 A resolution, molecular replacement and modeling using DHFR structure from Bacillus stearothermophilus, PDB ID 1ZDR, as search model	Enterococcus faecalis

Crystallization (Comment)

Organism

purified recombinant detagged enzyme in apoform or complex with inhibitor RAB, sitting drop vapor diffusion method, for the apoenzyme, mixing of 800 nl of 16.5 mg/ml protein in 20 mM Tris, pH 8.0, 150 mM NaCl, and 2 mM DTT, with 800 nl of well solution containing 5% PEG 3350 and 1.5 M ammonium citrate dibasic, pH 7.0, 1 week at room temperature, crystals complexed with RAB are grown from 1.1 M ammonium tartrate dibasic, pH 7.0, at 4°C in 2-3 months, X-ray diffraction structure determination and analysis at 2.1-2.3 A resolution, molecular replacement and modeling using DHFR structure from Bacillus stearothermophilus, PDB ID 1ZDR, as search model

Enterococcus faecalis

Protein Variants

Protein Variants	Comment	Organism
additional information	the C-terminal Strep-tag does not affect enzyme activity or inhibition by RAB-propyl inhibitors	Enterococcus faecalis

Inhibitors

Inhibitors	Comment	Organism
(2E)-1-(1-cyclohexylphthalazin-2(1H)-yl)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-phenoxyphthalazin-2(1H)-yl)prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-propylphthalazin-2(1H)-yl)prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(2-methylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3,3,3-trifluoropropyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3,5-dimethylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-fluorophenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-methylbut-2-en-1-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-methylbutyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-fluorophenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methoxyphenoxy)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methylphenoxy)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(hexan-3-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(propan-2-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-[4-(trifluoromethoxy)phenoxy]phthalazin-2(1H)-yl]prop-2-en-1-one	-	Enterococcus faecalis
additional information	design of an inhibitor series from the antifolate trimethoprim with a hydrophobic dihydrophthalazine heterocycle, and determination of the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid, overview. Four buried waters act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB binding. Substrate inhibitors may gain advantages in potency by the perturbation of cofactor dynamics. The inhibitors show competitive inhibition. Exogenous folate and low pH at pH 5.8 reduce the MIC values of the anti-folate inhibitors	Enterococcus faecalis
TMP	-	Enterococcus faecalis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
7,8-dihydrofolate + NADPH + H+	Enterococcus faecalis	-	5,6,7,8-tetrahydrofolate + NADP+	-	r
7,8-dihydrofolate + NADPH + H+	Enterococcus faecalis ATCC 29212	-	5,6,7,8-tetrahydrofolate + NADP+	-	r

Organism

Organism	UniProt	Comment	Textmining
Enterococcus faecalis	Q834R2	-	-
Enterococcus faecalis ATCC 29212	Q834R2	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally StrepII-tagged DHFR from Escherichia coli strain BL21(DE3)pLysS by affinity chromatography and tag cleavage with thrombin, followed by another step of affinity chromatography and gell filtration	Enterococcus faecalis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
7,8-dihydrofolate + NADPH + H+	-	Enterococcus faecalis	5,6,7,8-tetrahydrofolate + NADP+	-	r
7,8-dihydrofolate + NADPH + H+	-	Enterococcus faecalis ATCC 29212	5,6,7,8-tetrahydrofolate + NADP+	-	r

Synonyms

Synonyms	Comment	Organism
DHFR	-	Enterococcus faecalis

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	-	Enterococcus faecalis
NADPH	-	Enterococcus faecalis

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.0000021	-	TMP	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000026	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-phenylphthalazin-2(1H)-yl)prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000027	-	(2E)-1-(1-cyclohexylphthalazin-2(1H)-yl)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000035	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-methylbutyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000038	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(propan-2-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000038	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3,3,3-trifluoropropyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000038	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-methylbut-2-en-1-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000045	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-propylphthalazin-2(1H)-yl)prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000048	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3-fluorophenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.000005	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-(1-phenoxyphthalazin-2(1H)-yl)prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000051	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000056	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-fluorophenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000062	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methylphenoxy)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000067	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(4-methoxyphenoxy)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000071	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(3,5-dimethylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000072	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(2-methylphenyl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000074	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-(hexan-3-yl)phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis
0.0000188	-	(2E)-3-[5-[(2,4-diaminopyrimidin-5-yl)methyl]-2,3-dimethoxyphenyl]-1-[1-[4-(trifluoromethoxy)phenoxy]phthalazin-2(1H)-yl]prop-2-en-1-one	pH and temperature not specified in the publication	Enterococcus faecalis

General Information

General Information	Comment	Organism
additional information	role for water in stabilizing the DHFR substrate pocket and of Cys52, overview	Enterococcus faecalis