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(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
(S)-pantoate + NAD+
2-dehydropantoate + NADH + H+
low activity
-
-
r
(S)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
low activity
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
2-keto-3-hydroxyisovalerate + NADPH
? + NADP+
2-oxoisovalerate + NADPH
2-hydroxyvalerate + NADP+
-
low activity
-
-
r
2-oxopantoate + 3'-NADPH
(R)-pantoate + 3'-NADP+
-
-
-
-
r
2-oxopantoate + alpha-NADPH
(R)-pantoate + alpha-NADP+
-
-
-
-
r
2-oxopantoate + beta-NADPH
(R)-pantoate + beta-NADP+
-
highly specific for
-
-
r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
2-oxopantoate + thio-NADPH
(R)-pantoate + thio-NADP+
-
-
-
-
r
3-methyl-2-oxo-n-valerate + NADPH
2-hydroxy-3-methyl-n-valerate + NADP+
-
low activity
-
-
r
alpha-ketopantoate + NADPH
D-pantoate + NADP+
ketopantoate + ?
pantothenate + ?
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
ketopantoic acid + ?
D-pantoic acid
ketopantoic acid + NADH
pantoic acid + NAD+
-
-
-
?
ketopantoic acid + NADPH
D-pantoic acid + NADP+
ketopantoic acid + NADPH
pantoic acid + NADP+
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
additional information
?
-
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
-
-
-
r
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
substrate binding structure analysis, overview
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
substrate binding structure analysis, overview
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
-
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
the TK1968 protein displays reductase activity specific for 2-oxopantoate and prefers NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
-
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
the TK1968 protein displays reductase activity specific for 2-oxopantoate and prefers NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
-
-
-
?
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
an essential step for the biosynthesis of pantothenate, i.e. vitamin B5
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
the enzyme is involved in the biosynthesis of pantothenate, i.e. vitamin B5
-
-
?
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
substrate and product binding structures, hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys176 to form a key hydrogen bond with the C2 hydroxyl of pantoate, pantoate forms additional interactions with conserved residues Ser244, Asn98, and Asn180 and with two conservatively varied residues, Asn194 and Asn241, overview
-
-
r
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
-
?
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
-
?
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
r
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
r
2-keto-3-hydroxyisovalerate + NADPH
? + NADP+
-
-
-
r
2-keto-3-hydroxyisovalerate + NADPH
? + NADP+
-
-
-
r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
-
-
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
-
reaction is part of the D-pantothenate biosynthesis
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
-
part of the pantothenate biosynthesis
-
-
?
alpha-ketopantoate + NADPH
D-pantoate + NADP+
-
-
-
r
alpha-ketopantoate + NADPH
D-pantoate + NADP+
-
B-specific
-
r
alpha-ketopantoate + NADPH
D-pantoate + NADP+
-
pantothenate/coenzyme A biosynthetic pathway
-
r
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
-
-
-
r
ketopantoate + NADPH
pantoate + NADP+
pantothenate biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
substrate binding structure and thermodynamics
-
-
r
ketopantoate + NADPH
pantoate + NADP+
-
the 4-proS hydrogen is transferred from NADPH to ketopantoate to form pantoate and NADP+, ligand binding analysis by NMR spectroscopy
-
-
r
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
thiamine synthesis, pantothenate and thiamine biosynthetic pathway
-
?
ketopantoic acid + ?
D-pantoic acid
-
-
-
?
ketopantoic acid + ?
D-pantoic acid
-
-
-
?
ketopantoic acid + ?
D-pantoic acid
-
-
-
?
ketopantoic acid + ?
D-pantoic acid
-
-
-
?
ketopantoic acid + NADPH
D-pantoic acid + NADP+
-
-
-
r
ketopantoic acid + NADPH
D-pantoic acid + NADP+
-
-
-
r
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
B-specific
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
B-specific
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
-
-
-
?
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
-
-
-
-
?
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
-
-
-
-
?
additional information
?
-
-
-
-
-
?
additional information
?
-
-
-
-
?
additional information
?
-
-
-
-
-
?
additional information
?
-
-
no activity for double mutant K176A/E256A
-
-
?
additional information
?
-
complex formation of 2'-monophosphoadenosine 5'-diphosphoribose upon incubation of NADPH at pH 5.0, structure analysis, overview
-
-
?
additional information
?
-
-
complex formation of 2'-monophosphoadenosine 5'-diphosphoribose upon incubation of NADPH at pH 5.0, structure analysis, overview
-
-
?
additional information
?
-
conformational changes can occur upon substrate binding in the hinge region leading to partial closure of the cleft between the domains. Such motions may be present to some degree in the apo form
-
-
?
additional information
?
-
-
-
-
-
?
additional information
?
-
-
-
-
-
?
additional information
?
-
-
activity with ketopantoic acid is 100%, with ketopantoyl lactone 6%, with oxoalacetic acid 4%, no activity with 2-ketoisovaleric acid, pyruvic acid, 3-hydroxypyruvic acid, 3-phosphohydroxypyruvic acid, 2-ketobutyric acid, 2-ketoglutaric acid, and acetaldehyde
-
-
?
additional information
?
-
-
activity with ketopantoic acid is 100%, with ketopantoyl lactone 6%, with oxoalacetic acid 4%, no activity with 2-ketoisovaleric acid, pyruvic acid, 3-hydroxypyruvic acid, 3-phosphohydroxypyruvic acid, 2-ketobutyric acid, 2-ketoglutaric acid, and acetaldehyde
-
-
?
additional information
?
-
-
no activity with pantoate, ketoisovalerate, oxaloacetate, pyruvate, 3-hydroxypyruvic acid, alpha-ketoglutarate, alpha-ketobutyrate, acetaldehyde at 0.5 mM
-
-
?
additional information
?
-
-
3.8-fold higher specific activity with NADPH than with NADH
-
-
?
additional information
?
-
-
high specificity, among a variety of carbonyl compounds only ketopantoic acid and 2-keto-3-hydroxyisovalerate can serve as substrate
-
-
?
additional information
?
-
-
high specificity, among a variety of carbonyl compounds only ketopantoic acid and 2-keto-3-hydroxyisovalerate can serve as substrate
-
-
?
additional information
?
-
D-pantoate is much more preferred over L-pantoate in the reduction reaction, suggesting that the enzyme generates D-pantoate from 2-oxopantoate in the oxidation reaction
-
-
?
additional information
?
-
-
D-pantoate is much more preferred over L-pantoate in the reduction reaction, suggesting that the enzyme generates D-pantoate from 2-oxopantoate in the oxidation reaction
-
-
?
additional information
?
-
D-pantoate is much more preferred over L-pantoate in the reduction reaction, suggesting that the enzyme generates D-pantoate from 2-oxopantoate in the oxidation reaction
-
-
?
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(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
2-oxopantoate + NADPH
(R)-pantoate + NADP+
alpha-ketopantoate + NADPH
D-pantoate + NADP+
-
pantothenate/coenzyme A biosynthetic pathway
-
r
ketopantoate + NADPH
pantoate + NADP+
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
?
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
-
-
-
r
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
-
r
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
-
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
the TK1968 protein displays reductase activity specific for 2-oxopantoate and prefers NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
the TK1968 protein displays reductase activity specific for 2-oxopantoate and prefers NADH as the electron donor, distinct to the bacterial/eukaryotic NADPH-dependent enzymes
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
an essential step for the biosynthesis of pantothenate, i.e. vitamin B5
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
the enzyme is involved in the biosynthesis of pantothenate, i.e. vitamin B5
-
-
?
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
-
?
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
-
?
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
-
-
-
r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
-
reaction is part of the D-pantothenate biosynthesis
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
-
part of the pantothenate biosynthesis
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
pantothenate biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
-
thiamine synthesis, pantothenate and thiamine biosynthetic pathway
-
?
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
-
-
-
-
?
ketopantolactone + NADPH + H+
D-(-)-pantolactone + NADP+
-
-
-
-
?
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0.0096 - 8.1
(R)-4-dehydropantoate
0.006 - 0.03
2-dehydropantoate
8.55
2-Keto-3-hydroxyisovalerate
-
-
8.7
2-oxoisovalerate
-
pH 7.5, 25°C, recombinant enzyme
0.075
3'-NADPH
-
pH 7.5, 25°C, recombinant enzyme
3.8
3-methyl-2-oxo-n-valerate
-
pH 7.5, 25°C, recombinant enzyme
0.036
alpha-NADPH
-
pH 7.5, 25°C, recombinant enzyme
0.004
beta-NADPH
-
pH 7.5, 25°C, recombinant enzyme
52.1
D-pantoic acid
-
pH 8.0
0.4 - 0.742
ketopantoic acid
0.041
NAD+
recombinant enzyme, pH 6.4, 70°C
0.003
NADH
recombinant enzyme, pH 6.4, 70°C
0.018
thio-NADPH
-
pH 7.5, 25°C, recombinant enzyme
additional information
additional information
-
0.0096
(R)-4-dehydropantoate
wild type enzyme, at pH 7.5 and 25°C
8.1
(R)-4-dehydropantoate
mutant enzyme A181L, at pH 7.5 and 25°C
0.13
(R)-pantoate
recombinant enzyme, pH 6.4, 70°C
2.04
(R)-pantoate
recombinant enzyme, pH 6.4, 70°C
0.006
2-dehydropantoate
recombinant enzyme, pH 6.4, 70°C
0.03
2-dehydropantoate
pH 7.5, 27°C, wild-type enzyme
0.12
2-oxopantoate
-
pH 7.5, 25°C, recombinant enzyme
3.4
2-oxopantoate
-
30°C, pH 7.5
0.038
ketopantoate
-
+/-0.009, mutant K176C, alkylated
0.07
ketopantoate
-
+/-0.01, mutant K176C
0.12
ketopantoate
-
+/-0.008
0.95
ketopantoate
-
+/-0.29, mutant E256D
7.5
ketopantoate
-
+/-2.9, mutant K256A
40
ketopantoate
-
+/-6, mutant K176A
0.4
ketopantoic acid
-
pH 7.0
0.742
ketopantoic acid
-
+/-0.01
0.00135
NADPH
recombinant enzyme, pH 6.4, 70°C
0.002
NADPH
-
+/-0.0003, mutant E256A
0.0029
NADPH
-
+/-0.0006, mutant E256D
0.0038
NADPH
-
+/-0.0003, mutant K176C, alkylated
0.0057
NADPH
mutant enzyme A181L, at pH 7.5 and 25°C
0.0066
NADPH
-
+/-0.0016, mutant K176C
0.007
NADPH
pH 7.5, 27°C, wild-type enzyme
0.0072
NADPH
wild type enzyme, at pH 7.5 and 25°C
0.016
NADPH
-
+/-0.003, mutant K176A
additional information
additional information
-
steady-state kinetics, pH-dependence of kinetics with different substrates, overview
-
additional information
additional information
-
kinetics and thermodynamics, overview
-
additional information
additional information
kinetics and thermodynamics, wild-type enzyme, overview
-
additional information
additional information
-
kinetics and thermodynamics, wild-type enzyme, overview
-
additional information
additional information
kinetics of recombinant wild-type and mutant enzymes, overview
-
additional information
additional information
-
kinetics of recombinant wild-type and mutant enzymes, overview
-
additional information
additional information
Michaelis-Menten kinetics in both reaction directions
-
additional information
additional information
-
Michaelis-Menten kinetics in both reaction directions
-
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
-
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
-
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Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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evolution
the enzyme is a member of the family of 2-hydroxyacid dehydrogenases
malfunction
-
both the homologous gene from Enterococcus faecalis V583 (EF1861) and Escherichia coli panE functionally complement Francisella novicida lacking any KPR. Furthermore, panG from Francisella novicida can complement an Escherichia coli KPR double mutant. A panG deletion mutant is a pantothenate auxotroph and is genetically and chemically complemented with pang in trans or with the addition of pantolactone
malfunction
gene disruption of gene TK1968results in a strain with growth defects that are complemented by addition of pantoate
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
malfunction
-
gene disruption of gene TK1968results in a strain with growth defects that are complemented by addition of pantoate
-
metabolism
ketopantoate reductase from the hyperthermophilic archaeon Thermococcus kodakarensis catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. CoA biosynthesis in the archaeon is regulated by feedback inhibition of enzyme ketopantoate reductase
metabolism
-
ketopantoate reductase from the hyperthermophilic archaeon Thermococcus kodakarensis catalyses the second step in CoA biosynthesis, the reduction of 2-oxopantoate to pantoate. CoA biosynthesis in the archaeon is regulated by feedback inhibition of enzyme ketopantoate reductase
-
physiological function
ketopantoate reductase (KPR) catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, and is a target of the feedback inhibition by CoA in archaea. Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA
physiological function
ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A
physiological function
ketopantoate reductase catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate which is a step in the biosynthesis of coenzyme A
physiological function
-
ketopantoate reductase catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate which is a step in the biosynthesis of coenzyme A
-
physiological function
-
ketopantoate reductase (KPR) catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, and is a target of the feedback inhibition by CoA in archaea. Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA
-
additional information
CoA and 2-oxopantoate cooperatively trigger a conformational change from an open form to a closed enzyme form, structure analysis, overview
additional information
-
CoA and 2-oxopantoate cooperatively trigger a conformational change from an open form to a closed enzyme form, structure analysis, overview
additional information
residue Ser239 is known to be important for the binding affinity of 2-dehydropantoate
additional information
-
residue Ser239 is known to be important for the binding affinity of 2-dehydropantoate
additional information
-
CoA and 2-oxopantoate cooperatively trigger a conformational change from an open form to a closed enzyme form, structure analysis, overview
-
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prismic crystals, hanging drop vapor-diffusion technique
purified enzyme with bound NADP+, hanging drop vapour diffusion method, 10-15 mg/ml protein at 4°C is mixed with ketopantoate and NADP+ in a ratio of 5:1 and 2:1, respectively, in 0.1 M sodium acetate, pH 4.0-5.0, with 10%2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis at 2.1 A resolution, ternary complex modelling
purified recombinant His6-tagged enzyme in complex with 2'-monophosphoadenosine 5'-diphosphoribose, 4°C, 10-15 mg/ml protein with NADPH and pantoate at a final ligand:protein ratio of 2:1 and 5:1, respectively, mixing with 10% 2-methyl-2,4-pentanediol buffered with 0.1 M sodium acetate pH 4.0-5.0, X-ray diffraction structure determination and analysis at 1.95-2.0 A resolution
purified recombinant His6-tagged enzyme in complex with NADP+ and pantoate, hanging drop vapor-diffusion technique, 20°C, 15-30 mg/ml protein with 2 mM NADP+ and 10 mM pantoate, 0.002 ml of protein solution is mixed with an equal volume of well solution containing 35% v/v dioxane, cryoprotection with 20% v/v 2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis at 2.3 A resolution
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
sitting drop vapor diffusion method, using 300 mM magnesium acetate, 100 mM MES buffer (pH 6.6), and 15% (w/v) polyethylene glycol 3350
fine needle, at about 40% ammonium sulfate saturation
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purified enzyme in complex with CoA and 2-oxopantoate, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein and 1 mM CoA and 1 mM 2-oxopantoate with 0.001 ml of reservoir solution containing 100 mM Na acetate, pH 4.5, 20-25% v/v 2-methyl-2,4-pentanediol, and equilibration against 0.5 ml of reservoir solution, at 20°C, 3 days, X-ray diffraction structure determination and analysis at 1.65 A resolution, modeling by molecular replacement method using N-terminal (1-165 residues) and C-terminal (171-309 residues) domains of Ec-KPR structure, PDB ID 2OFP, as separated search models, respectively
purified recombinant tagged enzyme mutant C84A, hanging drop vapour diffusion method, mixing of 0.001 ml of 10 mg /ml protein in 10 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, mM NADH, and 1 mM 2-oxopantoate, with 0.001 ml of reservoir solution containing 100 mM sodium acetate, pH 5.5, 10% w/v PEG 3350, 20% v/v 2-propanol, and equilibration against 0.5 ml reservoir solution, at 20°C, 1 week, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement method for modeling
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E240A
-
site-directed mutagenesis, wild-type activity
E256D
-
wild-type activity
K176A/E256A
-
double mutant, no activity
K176C
-
wild-type activity
R31A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
W129A
site-directed mutagenesis
Y60A
site-directed mutagenesis
W129A
-
site-directed mutagenesis
-
Y60A
-
site-directed mutagenesis
-
D248A
-
site-directed mutagenesis, wild-type activity
D248A
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme, functional complementation of a panE knockout mutant strain
E210A
-
site-directed mutagenesis, wild-type activity
E210A
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme, functional complementation of a panE knockout mutant strain
E256A
-
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
E256A
site-directed mutagenesis, nearly inactive mutant, 2600fold decreased catalytic efficiency, no complementation of a panE knockout mutant strain
E256A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
K176A
-
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
K176A
site-directed mutagenesis, nearly inactive mutant, 78000fold decreased catalytic efficiency, no complementation of a panE knockout mutant strain
K176A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
K72A
-
site-directed mutagenesis, wild-type activity
K72A
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme, functional complementation of a panE knockout mutant strain
K72A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
N98A
site-directed mutagenesis, nearly inactive mutant, 4000fold reduced catalytic efficiency, no complementation of a panE knockout mutant strain
N98A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
S244A
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme, functional complementation of a panE knockout mutant strain
S244A
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
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
A181L
the substitution increases the Km of ketopantoate 844fold, without affecting the kcat value
C84A
site-directed mutagenesis
C84A
site-directed mutagenesis, crystal structure analysis, overview
C84A
-
site-directed mutagenesis
-
C84A
-
site-directed mutagenesis, crystal structure analysis, overview
-
additional information
-
Lys176 acts as general acid in ketopantoate reduction and is involved in catalysis and ketopantoate binding, E256A functions in D-pantoate and ketopantoate binding in ketopantoate reductase
additional information
Lys176 and Glu256 important for binding ketopantoate and the catalytic mechanism
additional information
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Lys176 and Glu256 important for binding ketopantoate and the catalytic mechanism
additional information
generation of a gene TK1968 disruption mutant
additional information
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generation of a gene TK1968 disruption mutant
additional information
-
generation of a gene TK1968 disruption mutant
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Novel enzymic production of D-(-)-pantoyl lactone through the stereospecific reduction of ketopantoic acid
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Escherichia coli
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Frodyma, M.E.; Downs, D.
ApbA, the ketopantoate reductase enzyme of Salmonella typhimurium is required for the synthesis of thiamine via the alternative pyrimidine biosynthetic pathway
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5572-5576
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Saccharomyces cerevisiae, Escherichia coli, Stenotrophomonas maltophilia, Salmonella enterica subsp. enterica serovar Typhimurium, Stenotrophomonas maltophilia 845, Escherichia coli BL21/lambdaDE3
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Kinetic and mechanistic analysis of the E. coli panE-encoded ketopantoate reductase
Biochemistry
39
3708-3717
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Escherichia coli
brenda
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Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue
Biochemistry
39
16244-16251
2000
Escherichia coli
brenda
Matak-Vinkovic, D.; Vinkovic, M.; Saldanha, S.A.; Ashurst, J.L.; von Delft, F.; Inoue, T.; Miguel, R.N.; Smith, A.G.; Blundell, T.L.; Abell, C.
Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism
Biochemistry
40
14493-14500
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Stenotrophomonas maltophilia, Salmonella enterica subsp. enterica serovar Typhimurium, Escherichia coli (P0A9J4), Escherichia coli, Stenotrophomonas maltophilia 845
brenda
Zheng, R.; Blanchard, J.S.
Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase
Biochemistry
42
11289-11296
2003
Escherichia coli
brenda
Merkamm, M.; Chassagnole, C.; Lindley, N.D.; Guyonvarch, A.
Ketopantoate reductase activity is only encoded by ilvC in Corynebacterium glutamicum
J. Biotechnol.
104
253-260
2003
Corynebacterium glutamicum
brenda
Ciulli, A.; Abell, C.
Biophysical tools to monitor enzyme-ligand interactions of enzymes involved in vitamin biosynthesis
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33
767-771
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Escherichia coli
brenda
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The crystal structure of Escherichia coli ketopantoate reductase with NADP+ bound
Biochemistry
44
8930-8939
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Escherichia coli (P0A9J4), Escherichia coli
brenda
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49
4992-5000
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Escherichia coli
brenda
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pH-tuneable binding of 2-phospho-ADP-ribose to ketopantoate reductase: a structural and calorimetric study
Acta Crystallogr. Sect. D
63
171-178
2007
Escherichia coli (P0A9J4), Escherichia coli
brenda
Ciulli, A.; Chirgadze, D.Y.; Smith, A.G.; Blundell, T.L.; Abell, C.
Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound: substrate recognition, conformational change, and cooperativity
J. Biol. Chem.
282
8487-8497
2007
Escherichia coli (P0A9J4), Escherichia coli
brenda
Headey, S.J.; Vom, A.; Simpson, J.S.; Scanlon, M.J.
Backbone assignments of the 34 kDa ketopantoate reductase from E. coli
Biomol. NMR Assign.
2
93-96
2008
Escherichia coli (P0A9J4)
brenda
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PanG, a new ketopantoate reductase involved in pantothenate synthesis
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195
965-976
2013
Francisella tularensis subsp. novicida
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The crystal structure of D-mandelate dehydrogenase reveals its distinct substrate and coenzyme recognition mechanisms from those of 2-ketopantoate reductase
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439
109-114
2013
Enterococcus faecalis (E3USM3), Enterococcus faecalis IAM10071 (E3USM3), Enterococcus faecalis IAM10071
brenda
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Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis
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90
307-321
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Thermococcus kodakarensis (Q5JGC2), Thermococcus kodakarensis, Thermococcus kodakarensis ATCC BAA-918 (Q5JGC2)
brenda
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Crystal structure of ketopantoate reductase from Thermococcus kodakarensis complexed with NADP(.)
Acta crystallogr. Sect. F
72
369-375
2016
Thermococcus kodakarensis (Q5JGC2), Thermococcus kodakarensis, Thermococcus kodakarensis ATCC BAA-918 (Q5JGC2)
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Evidence of kinetic cooperativity in dimeric ketopantoate reductase from Staphylococcus aureus
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54
3360-3369
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Staphylococcus aureus (A0A0J9X283), Staphylococcus aureus
brenda
Aikawa, Y.; Nishitani, Y.; Tomita, H.; Atomi, H.; Miki, K.
Crystal structure of archaeal ketopantoate reductase complexed with coenzyme a and 2-oxopantoate provides structural insights into feedback regulation
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84
374-382
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Thermococcus kodakarensis (Q5JGC2), Thermococcus kodakarensis, Thermococcus kodakarensis ATCC BAA-918 (Q5JGC2)
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Metabolic engineering of Escherichia coli for D-pantothenic acid production
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294
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Escherichia coli, Escherichia coli W3110
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Pei, X.; Wang, J.; Zheng, H.; Cheng, P.; Wu, Y.; Wang, A.; Su, W.
Highly efficient asymmetric reduction of ketopantolactone to D-(-)-pantolactone by Escherichia coli cells expressing recombinant conjugated polyketone reductase and glucose dehydrogenase in a fed-batch biphasic reaction system
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531-538
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Candida dubliniensis, Candida dubliniensis CD36
-
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