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Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.3 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

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

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 (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
1.5.1.3 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
1.5.1.3 TMP
-
 Enterococcus faecalis

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+ Enterococcus faecalis
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 r
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Enterococcus faecalis ATCC 29212
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Enterococcus faecalis Q834R2
-
-
1.5.1.3 Enterococcus faecalis ATCC 29212 Q834R2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Enterococcus faecalis 5,6,7,8-tetrahydrofolate + NADP+
-
 r
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Enterococcus faecalis ATCC 29212 5,6,7,8-tetrahydrofolate + NADP+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.5.1.3 DHFR
-
 Enterococcus faecalis

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADP+
-
 Enterococcus faecalis
1.5.1.3 NADPH
-
 Enterococcus faecalis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.5.1.3 0.0000021
-
 TMP pH and temperature not specified in the publication Enterococcus faecalis
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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
1.5.1.3 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

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