Information on EC 1.1.1.169 - 2-dehydropantoate 2-reductase and Organism(s) Escherichia coli and UniProt Accession P0A9J4

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This record set is specific for:
Escherichia coli
UNIPROT: P0A9J4


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


The taxonomic range for the selected organisms is: Escherichia coli

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
-
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Pantothenate and CoA biosynthesis
-
-
Metabolic pathways
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Biosynthesis of secondary metabolites
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-
SYSTEMATIC NAME
IUBMB Comments
(R)-pantoate:NADP+ 2-oxidoreductase
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CAS REGISTRY NUMBER
COMMENTARY hide
37211-74-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-dehydropantoate + NADPH
(R)-pantoate + 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
-
-
r
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
part of the pantothenate biosynthesis
-
-
?
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 + 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
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
part of the pantothenate biosynthesis
-
-
?
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
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preference for beta-NADPH as cofactor
NADP+
NADPH
thio-NADPH
-
-
additional information
-
NAD+/NADH are poor cofactors
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-phospho-ADP-ribose
-
competitive
2'-phospho-AMP
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competitive
ADP
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competitive
AMP
-
competitive
ATP
-
noncompetitive
NADP+
-
competitive versus NADPH, noncompetitive versus ketopantoate
Pantoate
-
noncompetitive versus ketopantoate and NADPH
phosphate
-
competitive
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bromoethylamine
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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
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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
0.03
2-dehydropantoate
-
pH 7.5, 27C, wild-type enzyme
8.7
2-oxoisovalerate
-
pH 7.5, 25C, recombinant enzyme
0.12
2-oxopantoate
-
pH 7.5, 25C, recombinant enzyme
0.075
3'-NADPH
-
pH 7.5, 25C, recombinant enzyme
3.8
3-methyl-2-oxo-n-valerate
-
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
0.038 - 40
ketopantoate
0.007
NADP+
-
+/-0.002
0.002 - 0.02
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
25
2-dehydropantoate
-
pH 7.5, 27C, wild-type enzyme
8 - 40
ketopantoate
25
NADPH
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pH 7.5, 27C, wild-type enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
27
phosphate
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pH 7.6
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
7.6
-
assay at
additional information
-
activity decreases with pH increase, reduction of ketopantoic acid
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
pH-dependence of kinetics with different substrates, modeling, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
27
-
assay at
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33800
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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
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anion-exchange chromatography, gel filtration chromatography
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FPLC, anion exchange chromatography, gel filtration
gel chromatography
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homogeneity in a yield of 20-60 mg from 3-6 g of cells
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native wild-type enzyme by anion exchange and adsorption chromatography, and gel filtration, recombinant His6-tagged enzyme by nickel affinity chromatography
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on nickel-nitrilotriacetic acid resin and by gel filtration, more than 98% pure
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli BL21(DE3)
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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 panE, expression of His6-tagged wild-type and mutant enzymes
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KPR expressed using a pRSETA vector in Escherichia coli strain BL21(DE3)C41
overexpression in strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E240A
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site-directed mutagenesis, wild-type activity
E256D
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wild-type activity
K176A/E256A
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double mutant, no activity
K176C
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wild-type activity
R31A
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site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, overview
additional information