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Literature summary extracted from

  • Heaslet, H.; Harris, M.; Fahnoe, K.; Sarver, R.; Putz, H.; Chang, J.; Subramanyam, C.; Barreiro, G.; Miller, J.
    Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim (2009), Proteins, 76, 706-717.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.3 expressed in Escherichia coli Staphylococcus aureus
1.5.1.3 expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (AI) Staphylococcus aureus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.5.1.3 crystal structure of the wild-type chromosomal DHFR from Staphylococcus aureus in complex with NADPH and trimethoprim is determined to 1.95 A resolution. The enzyme maintains the conserved fold of DHFR observed in other species with the active site core formed by an eight-stranded beta-sheet and four alpha-helices surrounding the core Staphylococcus aureus
1.5.1.3 purified recombinant wild-type and mutant enzymes in complex with inhibitor trimethoprim, hanging drop vapour diffusion method, 30.6 mg/ml wild-type enzyme protein mixed with 1 mM NADPH and 1 mM trimethoprim and incubated on ice for 3 h, mixing the protein 1:1 with a reservoir solution containing 30 mM citric acid/40 mM bis-tris propane pH 6.4, 13.3% PEG 3350, and 16.7% PEG 6000 and incubating at 22°C, hexagonal rod crystals form in 1-2 weeks, X-ray diffraction structure determination and analysis at 3.0 and 1.95 A resolution, respectively, molecular replacement and structure modelling Staphylococcus aureus

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.3 N48E/N130D site-directed mutagenesis, the S1 mutant enzyme shows improved expression levels and solubility. Inhibition kinetics and inhibitor binding thermodynamics in comparison to the wild-type enzyme, overview. In the absence of substrate and cofactor the active site of S1 DHFR is blocked, trimethoprim shows loss of potency and NADPH synergy on binding S1 DHFR Staphylococcus aureus
1.5.1.3 N48E/N130D/Y98F/A43G site-directed mutagenesis, inhibition kinetics and inhibitor binding thermodynamics in comparison to the wild-type enzyme, overview Staphylococcus aureus
1.5.1.3 Y98F/A43G inhibitor trimethoprim shows loss potency and NADPH synergy on binding S1 mutant DHFR. Mutation of residues Y98F/A43G in S1 mutant restores trimethoprim sensitivity and NADPH synergy Staphylococcus aureus

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.5.1.3 trimethoprim conformational changes in the Met20 loop on ligand binding, inhibitor binding thermodynamics, binding structure determined with wild-type and mutant enzymes, crystal structures, single type of independent enzyme binding site, overview Staphylococcus aureus

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining

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+ Staphylococcus aureus
-
 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.3 Staphylococcus aureus
-
-
-
1.5.1.3 Staphylococcus aureus P13955
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.3 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 (AI) by adsorption chromatography and gel filtration Staphylococcus aureus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.3 7,8-dihydrofolate + NADPH + H+
-
 Staphylococcus aureus 5,6,7,8-tetrahydrofolate + NADP+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.5.1.3 DHFR
-
 Staphylococcus aureus
1.5.1.3 dihydrofolate reductase
-
 Staphylococcus aureus

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.3 NADPH
-
 Staphylococcus aureus
1.5.1.3 NADPH the presence of NADPH greatly enhances binding of trimethoprim to DHFR Staphylococcus aureus

General Information

EC Number General Information Comment Organism
1.5.1.3 malfunction inhibition of DHFR leads to the depletion of tetrahydrofolate and eventual cell death Staphylococcus aureus
1.5.1.3 malfunction inhibitor trimethoprim shows loss potency and NADPH synergy on binding S1 mutant DHFR. Mutation of residues Y98F/A43G in S1 mutant restores trimethoprim sensitivity and NADPH synergy Staphylococcus aureus
1.5.1.3 metabolism DHFR is a critical enzyme in the maintenance of reduced folate pools used in the biosynthetic pathways of purines, thymidylate, methionine, glycine, pantothenic acid, and N-formyl-methionyl tRNA Staphylococcus aureus
1.5.1.3 physiological function DHFR is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. DHFR is a critical enzyme in the maintenance of reduced folate pools used in the biosynthetic pathways of purines, thymidylate, methionine, glycine, pantothenic acid, and N-formyl-methionyl tRNA Staphylococcus aureus