Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Staphylococcus aureus |
recombinant expression of C-terminally His-tagged wild-type and mutant enzymes | Staphylococcus aureus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type and mutant A181L enzymes, sitting drop vapor diffusion , for the wild-type enzyme complexed with 2-dehydropantoateand NADP+: mixing of 0.001 ml of 10 mg/mL protein in 4 mM 2-dehydropantoate, 4 mM NADP+, 50 mM NaCl, and 25 mM Tris, pH 8.0 with 0.001 ml of reservoir solution containing 300 mM magnesium acetate, 100 mM MES buffer, pH 6.6, and 15% PEG 3350, 2-3 days, 20°C, for the mutant enzyme A181L complexed with NADP+: mixing of 0.001ml of 10 mg/mL protein in 4 mM NADP+, 50 mM NaCl, and 25 mM Tris, pH 8.0, with 0.001 ml of the reservoir solution containing 6% tacsimate, 100 mM MES, pH 6, and 15% PEG 3350, 2-3 days, 20°C, X-ray diffraction structure determination and analysis at 1.81 and 2.62 A resolution, respectively | Staphylococcus aureus |
sitting drop vapor diffusion method, using 300 mM magnesium acetate, 100 mM MES buffer (pH 6.6), and 15% (w/v) polyethylene glycol 3350 | Staphylococcus aureus |
Protein Variants | Comment | Organism |
---|---|---|
A181L | site-directed mutagenesis, the substitution displaces Ser239 and increases the Km for ketopantoate 844fold, without affecting kcat. The decrease in 2-dehydropantoate affinity enhances the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity | Staphylococcus aureus |
A181L | the substitution increases the Km of ketopantoate 844fold, without affecting the kcat value | Staphylococcus aureus |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
(R)-4-dehydropantoate | - |
Staphylococcus aureus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | the enzyme shows strong positive cooperativity in kinetics. The enzyme follows a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path, with a small degree of cooperativity between subunits. The mechanism of Staphylococcus aureus KPR is distinct from those of previously described members of the family of 2-hydroxyacid dehydrogenases | Staphylococcus aureus | |
0.0057 | - |
NADPH | mutant enzyme A181L, at pH 7.5 and 25°C | Staphylococcus aureus | |
0.0072 | - |
NADPH | wild type enzyme, at pH 7.5 and 25°C | Staphylococcus aureus | |
0.0096 | - |
(R)-4-dehydropantoate | wild type enzyme, at pH 7.5 and 25°C | Staphylococcus aureus | |
8.1 | - |
(R)-4-dehydropantoate | mutant enzyme A181L, at pH 7.5 and 25°C | Staphylococcus aureus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-4-dehydropantoate + NADPH + H+ | Staphylococcus aureus | - |
(R)-pantoate + NADP+ | - |
? | |
2-dehydropantoate + NADPH + H+ | Staphylococcus aureus | - |
(R)-pantoate + NADP+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Staphylococcus aureus | A0A0J9X283 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged wild-type and mutant enzymes | Staphylococcus aureus |
TALON affinity resin column chromatography | Staphylococcus aureus |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(R)-pantoate + NADP+ = 2-dehydropantoate + NADPH + H+ | the enzyme follows a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path, with a small degree of cooperativity between subunits. The mechanism of Staphylococcus aureus KPR is distinct from those of previously described members of the family of 2-hydroxyacid dehydrogenases | Staphylococcus aureus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-4-dehydropantoate + NADPH + H+ | - |
Staphylococcus aureus | (R)-pantoate + NADP+ | - |
? | |
2-dehydropantoate + NADPH + H+ | - |
Staphylococcus aureus | (R)-pantoate + NADP+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
dimer | the KPR dimer is stable in solution | Staphylococcus aureus |
homodimer | x-ray crystallography | Staphylococcus aureus |
More | homology modeling of structure of the ternary KPR complex using the crystal structure of Staphylococcus aureus apo-KPR as a search model, PDB ID 3G17 | Staphylococcus aureus |
Synonyms | Comment | Organism |
---|---|---|
ketopantoate reductase | - |
Staphylococcus aureus |
KPR | - |
Staphylococcus aureus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Staphylococcus aureus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
16.4 | - |
NADPH | mutant enzyme A181L, at pH 7.5 and 25°C | Staphylococcus aureus | |
16.6 | - |
(R)-4-dehydropantoate | wild type enzyme, at pH 7.5 and 25°C | Staphylococcus aureus | |
16.8 | - |
NADPH | wild type enzyme, at pH 7.5 and 25°C | Staphylococcus aureus | |
18.2 | - |
(R)-4-dehydropantoate | mutant enzyme A181L, at pH 7.5 and 25°C | Staphylococcus aureus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Staphylococcus aureus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | - |
Staphylococcus aureus |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.27 | - |
(R)-4-dehydropantoate | wild type enzyme, at pH 7.5 and 25°C | Staphylococcus aureus |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is a member of the family of 2-hydroxyacid dehydrogenases | Staphylococcus aureus |
malfunction | enzyme mutation A181L causes substitution of Ser239 and increases the Km for ketopantoate 844fold, without affecting kcat. The decrease in 2-dehydropantoate affinity enhances the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity | Staphylococcus aureus |
additional information | residue Ser239 is known to be important for the binding affinity of 2-dehydropantoate | Staphylococcus aureus |
physiological function | ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A | Staphylococcus aureus |