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Literature summary extracted from

  • Sanchez, J.E.; Gross, P.G.; Goetze, R.W.; Walsh, R.M.; Peeples, W.B.; Wood, Z.A.
    Evidence of kinetic cooperativity in dimeric ketopantoate reductase from Staphylococcus aureus (2015), Biochemistry, 54, 3360-3369.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.169 expressed in Escherichia coli BL21(DE3) cells Staphylococcus aureus
1.1.1.169 recombinant expression of C-terminally His-tagged wild-type and mutant enzymes Staphylococcus aureus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.169 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
1.1.1.169 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

EC Number Protein Variants Comment Organism
1.1.1.169 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
1.1.1.169 A181L the substitution increases the Km of ketopantoate 844fold, without affecting the kcat value Staphylococcus aureus

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.169 (R)-4-dehydropantoate
-
 Staphylococcus aureus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.169 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
1.1.1.169 0.0057
-
 NADPH mutant enzyme A181L, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 0.0072
-
 NADPH wild type enzyme, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 0.0096
-
 (R)-4-dehydropantoate wild type enzyme, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 8.1
-
 (R)-4-dehydropantoate mutant enzyme A181L, at pH 7.5 and 25°C Staphylococcus aureus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.169 (R)-4-dehydropantoate + NADPH + H+ Staphylococcus aureus
-
 (R)-pantoate + NADP+
-
 ?
1.1.1.169 2-dehydropantoate + NADPH + H+ Staphylococcus aureus
-
 (R)-pantoate + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.169 Staphylococcus aureus A0A0J9X283
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.169 recombinant C-terminally His-tagged wild-type and mutant enzymes Staphylococcus aureus
1.1.1.169 TALON affinity resin column chromatography Staphylococcus aureus

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.1.169 (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
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.169 (R)-4-dehydropantoate + NADPH + H+
-
 Staphylococcus aureus (R)-pantoate + NADP+
-
 ?
1.1.1.169 2-dehydropantoate + NADPH + H+
-
 Staphylococcus aureus (R)-pantoate + NADP+
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.169 dimer the KPR dimer is stable in solution Staphylococcus aureus
1.1.1.169 homodimer x-ray crystallography Staphylococcus aureus
1.1.1.169 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

EC Number Synonyms Comment Organism
1.1.1.169 ketopantoate reductase
-
 Staphylococcus aureus
1.1.1.169 KPR
-
 Staphylococcus aureus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.169 25
-
 assay at Staphylococcus aureus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.169 16.4
-
 NADPH mutant enzyme A181L, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 16.6
-
 (R)-4-dehydropantoate wild type enzyme, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 16.8
-
 NADPH wild type enzyme, at pH 7.5 and 25°C Staphylococcus aureus
1.1.1.169 18.2
-
 (R)-4-dehydropantoate mutant enzyme A181L, at pH 7.5 and 25°C Staphylococcus aureus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.169 7.5
-
 assay at Staphylococcus aureus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.169 NADPH
-
 Staphylococcus aureus

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.1.1.169 0.27
-
 (R)-4-dehydropantoate wild type enzyme, at pH 7.5 and 25°C Staphylococcus aureus

General Information

EC Number General Information Comment Organism
1.1.1.169 evolution the enzyme is a member of the family of 2-hydroxyacid dehydrogenases Staphylococcus aureus
1.1.1.169 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
1.1.1.169 additional information residue Ser239 is known to be important for the binding affinity of 2-dehydropantoate Staphylococcus aureus
1.1.1.169 physiological function ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A Staphylococcus aureus