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

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
Biosynthesis of secondary metabolites
-
-
Metabolic pathways
-
-
Pantothenate and CoA biosynthesis
-
-
pantothenate biosynthesis
-
-
phosphopantothenate biosynthesis I
-
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phosphopantothenate biosynthesis III
-
-
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
Escherichia coli BL21/lambdaDE3
BL21/lambdaDE3
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
NRRL Y-2034
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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-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
-
-
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
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
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
thio-NADPH
-
-
additional information
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
-
competitive
ADP
-
competitive
AMP
-
competitive
ATP
-
noncompetitive
Barbital
-
9.0% at 1 mM
D-pantoic acid
-
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
p-chloromercuribenzoate
-
sulfhydryl reagents, slightly inhibitory
Pantoate
-
noncompetitive versus ketopantoate and NADPH
phenylhydrazine
-
carbonyl reagent
phosphate
-
competitive
quercetin
-
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
-
activation of enzyme from K176A mutant
formate
-
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
-
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
8.55
2-Keto-3-hydroxyisovalerate
-
-
8.7
2-oxoisovalerate
-
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
-
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
0.007
NADP+
-
+/-0.002
0.002 - 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
25
2-dehydropantoate
Escherichia coli
-
pH 7.5, 27C, wild-type enzyme
8 - 40
ketopantoate
25
NADPH
Escherichia coli
-
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
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
7.5
-
assay at
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
25
-
assay at
27
-
assay at
35 - 40
37
-
reduction of ketopantoic acid
42
-
reduction of ketopantoic acid at pH 6.0
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 60
-
15C: about 70% of activity maximum, 60C: about 85% of activity maximum
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Cupriavidus pinatubonensis (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)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33800
-
calculated from 912 bp coding region
87000
-
+/-5000, sedimentation equilibrium method
115000
-
+/-5000, gel filtration
140000 - 160000
-
+/-5000, HPLC gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
oligomer
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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fine needle, at about 40% ammonium sulfate saturation
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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
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
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli BL21(DE3)
-
expression in Escherichia coli BL21/lambdaDE3
-
expression in Escherichia coli K12
-
expression of wild-type and mutant enzymes
-
gene ilvC, complementation of an enzyme-deficient Escherichia coli mutant strain
-
gene panE, expression of His6-tagged wild-type and mutant enzymes
-
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
additional information
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