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Literature summary extracted from

  • Lamb, K.M.; Lombardo, M.N.; Alverson, J.; Priestley, N.D.; Wright, D.L.; Anderson, A.C.
    Crystal structures of Klebsiella pneumoniae dihydrofolate reductase bound to propargyl-linked antifolates reveal features for potency and selectivity (2014), Antimicrob. Agents Chemother., 58, 7484-7491 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3) Klebsiella pneumoniae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.3 purified recombinant His-tagged enzyme in complex with NADPH and inhibitor 3 or 4, sitting drop vapour diffusion method, mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 4 and concentration to 24 mg/ml protein, addition of reservoir solution containing 100 mM cacodylate, pH 7.4, 150 mM sodium acetate, 25% w/v PEG 8000 in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 1 mM (final concentration) L-glutathione, or mixing of 15 mg/ml protein with 1 mM NADPH, and 1 mM inhibitor 3 and concentration to 24 mg/ml protein, addition of reservoir solution, containing 100 mM cacodylate, pH 7.4, 125 mM sodium acetate, 25% w/v PEG 8000, and 11 mM calcium chloride, in a 1:2 ratio to 0.01 ml sitting drops, equilibration against 1 ml reservoir solution with additional 11 mM (final concentration) betaine hydrochloride, both 1-5 days at 4°C, X-ray diffraction structure determination and analysis at 1.76 A resolution, molecular replacement Klebsiella pneumoniae

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
 Homo sapiens
1.5.1.3 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
 Klebsiella pneumoniae
1.5.1.3 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
 Homo sapiens
1.5.1.3 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
-
 Klebsiella pneumoniae
1.5.1.3 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
 Homo sapiens
1.5.1.3 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
 Klebsiella pneumoniae
1.5.1.3 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
-
 Homo sapiens
1.5.1.3 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine active site binding structure, overview Klebsiella pneumoniae
1.5.1.3 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
-
 Homo sapiens
1.5.1.3 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine active site binding structure, overview Klebsiella pneumoniae
1.5.1.3 additional information development of a series of propargyl-linked antifolates that are potent inhibitors of DHFR from multiple species. One of the strengths of this series is the flexibility and versatility to target TMP-resistant species, regardless of whether that resistance occurs natively or via acquired mutations. Determination and comparison of inhibition kinetics and binding structures of human and Klebsiella enzymes with propargyl-linked antifolate inhibitors, overview Homo sapiens
1.5.1.3 additional information development of a series of propargyl-linked antifolates that are potent inhibitors of DHFR from multiple species. One of the strengths of this series is the flexibility and versatility to target TMP-resistant species, regardless of whether that resistance occurs natively or via acquired mutations. Determination and comparison of inhibition kinetics and binding structures of human and Klebsiella enzymes with propargyl-linked antifolate inhibitors, overview Klebsiella pneumoniae
1.5.1.3 trimethoprim resistance of enterobacteria generally occurs through the acquisition of a TMP-insensitive DHFR Klebsiella pneumoniae
1.5.1.3 trimethoprim-sulfamethoxazole resistance of enterobacteria generally occurs through the acquisition of a TMP-insensitive DHFR Klebsiella pneumoniae

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+ Homo sapiens
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 r
1.5.1.3 7,8-dihydrofolate + NADPH + H+ Klebsiella pneumoniae
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Homo sapiens P00374
-
-
1.5.1.3 Klebsiella pneumoniae Q7BPB0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Klebsiella pneumoniae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Homo sapiens 5,6,7,8-tetrahydrofolate + NADP+
-
 r
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Klebsiella pneumoniae 5,6,7,8-tetrahydrofolate + NADP+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.5.1.3 DHFR
-
 Homo sapiens
1.5.1.3 DHFR
-
 Klebsiella pneumoniae

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.3 22
-
 assay at room temperature Homo sapiens
1.5.1.3 22
-
 assay at room temperature Klebsiella pneumoniae

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.3 7
-
 assay at Homo sapiens
1.5.1.3 7
-
 assay at Klebsiella pneumoniae

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADP+
-
 Homo sapiens
1.5.1.3 NADP+
-
 Klebsiella pneumoniae
1.5.1.3 NADPH
-
 Homo sapiens
1.5.1.3 NADPH
-
 Klebsiella pneumoniae

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
1.5.1.3 0.000015
-
 pH 7.0, 22°C Klebsiella pneumoniae 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
1.5.1.3 0.000016
-
 pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.000023
-
 pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.000058
-
 pH 7.0, 22°C Klebsiella pneumoniae 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
1.5.1.3 0.000108
-
 pH 7.0, 22°C Klebsiella pneumoniae 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.00016
-
 pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[3-methoxy-5-(pyrimidin-5-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.00033
-
 pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[2-methoxy-5-(pyridin-4-yl)phenyl]prop-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.00075
-
 pH 7.0, 22°C Homo sapiens 5-[3-(5-methoxy-3',5'-dimethyl[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine
1.5.1.3 0.0013
-
 pH 7.0, 22°C Homo sapiens 6-ethyl-5-[3-[3-methoxy-5-(pyridin-4-yl)phenyl]but-1-yn-1-yl]pyrimidine-2,4-diamine
1.5.1.3 0.0017
-
 pH 7.0, 22°C Homo sapiens 5-[3-(5-methoxy[1,1'-biphenyl]-3-yl)but-1-yn-1-yl]-6-methylpyrimidine-2,4-diamine