Information on EC 1.1.1.169 - 2-dehydropantoate 2-reductase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea

EC NUMBER
COMMENTARY hide
1.1.1.169
-
RECOMMENDED NAME
GeneOntology No.
2-dehydropantoate 2-reductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-pantoate + NADP+ = 2-dehydropantoate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
phosphopantothenate biosynthesis I
-
-
phosphopantothenate biosynthesis III (archaebacteria)
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-
pantothenate biosynthesis
-
-
Pantothenate and CoA biosynthesis
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-
Metabolic pathways
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
(R)-pantoate:NADP+ 2-oxidoreductase
-
CAS REGISTRY NUMBER
COMMENTARY hide
37211-74-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
soil isolates
-
-
Manually annotated by BRENDA team
gene ilvC, the enzyme activity in the organism is only encoded by ilvC, a bifunctional enzyme showing acetohydroxyacid isomeroreductase and 2-hydropantoate 2-reductase activity
-
-
Manually annotated by BRENDA team
BL21/lambdaDE3
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme is a member of the family of 2-hydroxyacid dehydrogenases
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
show the reaction diagram
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
show the reaction diagram
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
show the reaction diagram
(S)-pantoate + NAD+
2-dehydropantoate + NADH + H+
show the reaction diagram
low activity
-
-
r
(S)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
show the reaction diagram
low activity
-
-
r
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
show the reaction diagram
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
show the reaction diagram
2-keto-3-hydroxyisovalerate + NADPH
? + NADP+
show the reaction diagram
2-oxoisovalerate + NADPH
2-hydroxyvalerate + NADP+
show the reaction diagram
-
low activity
-
-
r
2-oxopantoate + 3'-NADPH
(R)-pantoate + 3'-NADP+
show the reaction diagram
-
-
-
-
r
2-oxopantoate + alpha-NADPH
(R)-pantoate + alpha-NADP+
show the reaction diagram
-
-
-
-
r
2-oxopantoate + beta-NADPH
(R)-pantoate + beta-NADP+
show the reaction diagram
-
highly specific for
-
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r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
2-oxopantoate + thio-NADPH
(R)-pantoate + thio-NADP+
show the reaction diagram
-
-
-
-
r
3-methyl-2-oxo-n-valerate + NADPH
2-hydroxy-3-methyl-n-valerate + NADP+
show the reaction diagram
-
low activity
-
-
r
alpha-ketopantoate + NADPH
D-pantoate + NADP+
show the reaction diagram
ketopantoate + ?
pantothenate + ?
show the reaction diagram
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
ketopantoic acid + ?
D-pantoic acid
show the reaction diagram
ketopantoic acid + NADH
pantoic acid + NAD+
show the reaction diagram
-
-
-
?
ketopantoic acid + NADPH
D-pantoic acid + NADP+
show the reaction diagram
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-4-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
show the reaction diagram
(R)-pantoate + NAD+
2-dehydropantoate + NADH + H+
show the reaction diagram
(R)-pantoate + NADP+
2-dehydropantoate + NADPH + H+
show the reaction diagram
2-dehydropantoate + NADH + H+
(R)-pantoate + NAD+
show the reaction diagram
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
2-dehydropantoate + NADPH + H+
(R)-pantoate + NADP+
show the reaction diagram
A0A0J9X283
-
-
-
r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
alpha-ketopantoate + NADPH
D-pantoate + NADP+
show the reaction diagram
-
pantothenate/coenzyme A biosynthetic pathway
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3'-NADPH
-
-
alpha-NADPH
-
-
beta-NADPH
-
preference for beta-NADPH as cofactor
NADP+
thio-NADPH
-
-
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(R)-4-dehydropantoate
-
2'-phospho-ADP-ribose
-
competitive
2'-phospho-AMP
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competitive
ADP
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competitive
AMP
-
competitive
ATP
-
noncompetitive
Barbital
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9.0% at 1 mM
D-pantoic acid
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noncompetitive with ketopantoic acid at saturation level of NADPH, noncompetitive with NADPH at unsaturation level of ketopantoic acid
Diphenylhydantoin
-
22.6% at 1 mM
iodoacetate
-
sulfhydryl reagents, slightly inhibitory
NaAsO2
-
carbonyl reagent
NADP+
p-chloromercuribenzoate
-
sulfhydryl reagents, slightly inhibitory
Pantoate
-
noncompetitive versus ketopantoate and NADPH
phenylhydrazine
-
carbonyl reagent
phosphate
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competitive
quercetin
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12.0% at 0.1 mM
Semicarbazide
-
carbonyl reagent
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bromoethylamine
-
activation of enzyme from K176C mutant
ethylamine
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activation of enzyme from K176A mutant
formate
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E256A mutant activity greatly increased, twice as high at pH 7.2 as at pH 5.9
methylamine
-
activation of enzyme from K176A mutant
Propylamine
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activation of enzyme from K176A mutant
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0096 - 8.1
(R)-4-dehydropantoate
0.13 - 2.04
(R)-pantoate
0.006 - 0.03
2-dehydropantoate
8.55
2-Keto-3-hydroxyisovalerate
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-
8.7
2-oxoisovalerate
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pH 7.5, 25C, recombinant enzyme
0.12 - 3.4
2-oxopantoate
0.075
3'-NADPH
-
pH 7.5, 25C, recombinant enzyme
3.8
3-methyl-2-oxo-n-valerate
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pH 7.5, 25C, recombinant enzyme
0.036
alpha-NADPH
-
pH 7.5, 25C, recombinant enzyme
0.004
beta-NADPH
-
pH 7.5, 25C, recombinant enzyme
52.1
D-pantoic acid
-
pH 8.0
0.038 - 40
ketopantoate
0.4 - 0.742
ketopantoic acid
0.041
NAD+
recombinant enzyme, pH 6.4, 70C
0.003
NADH
recombinant enzyme, pH 6.4, 70C
0.007
NADP+
-
+/-0.002
0.00135 - 0.776
NADPH
0.26
Pantoate
-
+/-0.04
0.018
thio-NADPH
-
pH 7.5, 25C, recombinant enzyme
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.6 - 18.2
(R)-4-dehydropantoate
3.17 - 3.8
(R)-pantoate
23.1 - 25
2-dehydropantoate
8 - 40
ketopantoate
2.41
NAD+
recombinant enzyme, pH 6.4, 70C
19.7
NADH
recombinant enzyme, pH 6.4, 70C
2.22 - 25
NADPH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 29
(R)-pantoate
3830
2-dehydropantoate
recombinant enzyme, pH 6.4, 70C
60
NAD+
recombinant enzyme, pH 6.4, 70C
6540
NADH
recombinant enzyme, pH 6.4, 70C
2220
NADPH
recombinant enzyme, pH 6.4, 70C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.27
(R)-4-dehydropantoate
wild type enzyme, at pH 7.5 and 25C
0.2
2'-phospho-ADP-ribose
-
pH 7.6
0.24
2'-phospho-AMP
-
pH 7.6
1.05
ADP
-
pH 7.6
6.3
AMP
-
pH 7.6
0.61
ATP
-
pH 7.6
0.000042
CoA
recombinant enzyme, pH 6.4, 70C
27
phosphate
-
pH 7.6
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0126
-
wild-type strain
0.0246
-
recombinant Escherichia coli mutant strain expresing the enzyme
0.38
-
at pH 6.5 using NADPH and ketopantoic acid
16
-
optimal reaction conditions using NADPH
61
-
optimal reaction conditions using NADH
633
-
crystalline enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
reduction of ketopantoic acid with NADPH
5.5
-
reduction of ketopantoic acid with NADH
6
-
reduction of ketopantoic acid
6.3
-
reduction of pantoic acid at 42C
7.6
-
assay at
8.5
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oxidation of D-pantoic acid
additional information
-
activity decreases with pH increase, reduction of ketopantoic acid
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.3 - 6.5
-
pH 4.3: about 10% of activity maximum, pH 6.5: about 40% of activity maximum
additional information
-
pH-dependence of kinetics with different substrates, modeling, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27
-
assay at
35 - 40
37
-
reduction of ketopantoic acid
42
-
reduction of ketopantoic acid at pH 6.0
90
recombinant enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 60
-
15C: about 70% of activity maximum, 60C: about 85% of activity maximum
50 - 110
and above, activity range, profile overview
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Cupriavidus necator (strain JMP 134 / LMG 1197)
Enterococcus faecalis (strain ATCC 700802 / V583)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Geobacter metallireducens (strain GS-15 / ATCC 53774 / DSM 7210)
Methylococcus capsulatus (strain ATCC 33009 / NCIMB 11132 / Bath)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Ralstonia solanacearum (strain Po82)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30500
-
3-5 * 30500 or 30000, SDS-PAGE
33800
-
calculated from 912 bp coding region
33866
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1 * 33000 gel filtration, 1 * 33866 elelectrospray mass spectrometry analysis, 1 * 33870 amino acid sequence
33870
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1 * 33000 gel filtration, 1 * 33866 elelectrospray mass spectrometry analysis, 1 * 33870 amino acid sequence
34048
2 * 34048, sequence calculation, 2 x 30000, about, recombinant enzyme, SDS-PAGE
60000
recombinant enzyme, gel filtration
87000
-
+/-5000, sedimentation equilibrium method
115000
-
+/-5000, gel filtration
140000 - 160000
-
+/-5000, HPLC gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
oligomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
side-chain modification
SeMet substitution
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
prismic crystals, hanging drop vapor-diffusion technique
purified enzyme with bound NADP+, hanging drop vapour diffusion method, 10-15 mg/ml protein at 4C 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
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purified recombinant His6-tagged enzyme in complex with 2'-monophosphoadenosine 5'-diphosphoribose, 4C, 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
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purified recombinant His6-tagged enzyme in complex with NADP+ and pantoate, hanging drop vapor-diffusion technique, 20C, 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
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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, 20C, 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, 20C, 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 20C, 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
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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 20C, 1 week, X-ray diffraction structure determination and analysis at 2.3 A resolution, molecular replacement method for modeling
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
-
30C, 30 min
286103
7
-
stable below 60C for 10 min
286103
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
30 min, pH 6-10
60
-
10 min, stable below 60C
60 - 90
purified recombinant enzyme, pH 6.4, completely stable at for at least 24 h
70
-
10 min, 70% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 0.01 M Tris/HCl buffer, pH 7.4, 0.1 mM DTT, 0.2 M NaCl, 6 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion-exchange chromatography, gel filtration chromatography
-
FPLC, anion exchange chromatography, gel filtration
gel chromatography
homogeneity in a yield of 20-60 mg from 3-6 g of cells
-
native wild-type enzyme by anion exchange and adsorption chromatography, and gel filtration, recombinant His6-tagged enzyme by nickel affinity chromatography
-
on nickel-nitrilotriacetic acid resin and by gel filtration, more than 98% pure
recombinant C-terminally His-tagged wild-type and mutant enzymes; TALON affinity resin column chromatography
recombinant enzyme from Escherichia coli by heat treatment, anion-exchange chromatography, and gel filtration
recombinant wild-type and mutant enzymes from Escherichia coli strain Rosetta2 (DE3) pLysS by nickel affinity chromatography, avidin affinity chromatography, and dialysis
-
recombinant wild-type and mutant Tk-KPR enzyme dimers carrying a His6-tag and a Strep-tag on each monomer from Escherichia coli by affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells; recombinant expression of C-terminally His-tagged wild-type and mutant enzymes
expression in Escherichia coli BL21(DE3)
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expression in Escherichia coli BL21/lambdaDE3
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expression in Escherichia coli K12
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expression of wild-type and mutant enzymes
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gene ilvC, complementation of an enzyme-deficient Escherichia coli mutant strain
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gene panE, expression of His6-tagged wild-type and mutant enzymes
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gene TK1968, DNA and amino acid sequence determination and analysis, sequence comparison, recombinant expression in Escherichia coli
gene TK1968, recombinant coexpression of wild-type and mutant Tk-KPR enzyme dimers carrying a His6-tag and a Strep-tag on each monomer in Escherichia coli
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gene TK1968, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain Rosetta2 (DE3) pLysS
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KPR expressed using a pRSETA vector in Escherichia coli strain BL21(DE3)C41
overexpression in strain BL21(DE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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; the substitution increases the Km of ketopantoate 844fold, without affecting the kcat value
W129A
-
site-directed mutagenesis
Y60A
-
site-directed mutagenesis
C84A
-
site-directed mutagenesis; site-directed mutagenesis, crystal structure analysis, overview
-
W129A
-
site-directed mutagenesis
-
Y60A
-
site-directed mutagenesis
-
additional information
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