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Literature summary for 1.1.1.169 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)

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 (Commentary)

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

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

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

KM Value [mM]

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Staphylococcus aureus A0A0J9X283
-
-

Purification (Commentary)

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

Reaction

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 and Products (Substrate)

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

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

Synonyms Comment Organism
ketopantoate reductase
-
Staphylococcus aureus
KPR
-
Staphylococcus aureus

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

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

Cofactor

Cofactor Comment Organism Structure
NADPH
-
Staphylococcus aureus

Ki Value [mM]

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

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