Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli | Staphylococcus aureus |
expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (AI) | Staphylococcus aureus |
Crystallization (Comment) | Organism |
---|---|
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 |
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 | Comment | Organism |
---|---|---|
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 |
N48E/N130D/Y98F/A43G | site-directed mutagenesis, inhibition kinetics and inhibitor binding thermodynamics in comparison to the wild-type enzyme, overview | Staphylococcus aureus |
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 | Comment | Organism | Structure |
---|---|---|---|
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 | Comment | Organism | GeneOntology No. | Textmining |
---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Staphylococcus aureus | - |
- |
- |
Staphylococcus aureus | P13955 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 (AI) by adsorption chromatography and gel filtration | Staphylococcus aureus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
7,8-dihydrofolate + NADPH + H+ | - |
Staphylococcus aureus | 5,6,7,8-tetrahydrofolate + NADP+ | - |
? |
Synonyms | Comment | Organism |
---|---|---|
DHFR | - |
Staphylococcus aureus |
dihydrofolate reductase | - |
Staphylococcus aureus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | - |
Staphylococcus aureus | |
NADPH | the presence of NADPH greatly enhances binding of trimethoprim to DHFR | Staphylococcus aureus |
General Information | Comment | Organism |
---|---|---|
malfunction | inhibition of DHFR leads to the depletion of tetrahydrofolate and eventual cell death | Staphylococcus aureus |
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 |
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 |
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 |