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
1.1.1.169
-
RECOMMENDED NAME
GeneOntology No.
2-dehydropantoate 2-reductase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
(R)-pantoate + NADP+ = 2-dehydropantoate + NADPH + H+
show the reaction diagram
-
-
-
-
(R)-pantoate + NADP+ = 2-dehydropantoate + NADPH + H+
show the reaction diagram
molecular catalytic mechanism, substrate and cofactor binding, Asn98, Glu256, and Lys176 are essential, overview
-
(R)-pantoate + NADP+ = 2-dehydropantoate + NADPH + H+
show the reaction diagram
protein-ligand interactions, overview
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Metabolic pathways
-
Pantothenate and CoA biosynthesis
-
phosphopantothenate biosynthesis I
-
phosphopantothenate biosynthesis III
-
SYSTEMATIC NAME
IUBMB Comments
(R)-pantoate:NADP+ 2-oxidoreductase
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2-ketopantoate reductase
-
-
-
-
2-ketopantoic acid reductase
-
-
-
-
2-oxopantoate reductase
-
-
-
-
ketopantoate reductase
-
-
-
-
ketopantoate reductase
-
-
ketopantoate reductase
-
-
ketopantoic acid reductase
-
-
-
-
KPA reductase
-
-
-
-
KPR
-
-
-
-
additional information
-
gene ilvC encodes a acetohydroxyacid isomeroreductase which also shows 2-hydropantoate 2-reductase activity
additional information
-
KPR belongs to the 6-phosphogluconate dehydrogenase superfamily in the SCOP database
CAS REGISTRY NUMBER
COMMENTARY
37211-74-8
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
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
ATCC 9637
-
-
Manually annotated by BRENDA team
BL21/lambdaDE3
-
-
Manually annotated by BRENDA team
expressed in BL21(DE3)
-
-
Manually annotated by BRENDA team
expressed in BL21(DE3)
Uniprot
Manually annotated by BRENDA team
K12, expressed in BL21(DE3)
-
-
Manually annotated by BRENDA team
K12, mutant completely lacking ketopantoate reductase activity
-
-
Manually annotated by BRENDA team
Escherichia coli BL21/lambdaDE3
BL21/lambdaDE3
-
-
Manually annotated by BRENDA team
Saccharomyces cerevisiae NRRL Y-2034
NRRL Y-2034
-
-
Manually annotated by BRENDA team
Stenotrophomonas maltophilia 845
845
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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 ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
-
-
-
?
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
an essential step for the biosynthesis of pantothenate, i.e. vitamin B5
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in the biosynthesis of pantothenate, i.e. vitamin B5
-
-
?
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
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-keto-3-hydroxyisovalerate + NADPH
? + NADP+
show the reaction diagram
Stenotrophomonas maltophilia, Stenotrophomonas maltophilia 845
-
-
-
r
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 + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
part of the pantothenate biosynthesis
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
reaction is part of the D-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
-
-
-
r
alpha-ketopantoate + NADPH
D-pantoate + NADP+
show the reaction diagram
-
B-specific, pantothenate/coenzyme A biosynthetic pathway
-
r
ketopantoate + ?
pantothenate + ?
show the reaction diagram
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
-
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
thiamine synthesis, pantothenate and thiamine biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-, P0A9J4
pantothenate biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
P0A9J4
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
P0A9J4
substrate binding structure and thermodynamics
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
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+
show the reaction diagram
Escherichia coli BL21/lambdaDE3
-
-
-
?
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
Stenotrophomonas maltophilia, Stenotrophomonas maltophilia 845
-
-
-
r
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
-
B-specific
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
Escherichia coli BL21/lambdaDE3
-
-
-
?
ketopantoic acid + NADPH
pantoic acid + NADP+
show the reaction diagram
Saccharomyces cerevisiae NRRL Y-2034
-
-
-
?
additional information
?
-
-
-
-
-
-
additional information
?
-
P0A9J4
-
-
-
-
additional information
?
-
-
-
-
-
-
additional information
?
-
-
high specificity, among a variety of carbonyl compounds only ketopantoic acid and 2-keto-3-hydroxyisovalerate can serve as substrate
-
-
-
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 for double mutant K176A/E256A
-
-
-
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
?
-
-
complex formation of 2'-monophosphoadenosine 5'-diphosphoribose upon incubation of NADPH at pH 5.0, structure analysis, overview
-
-
-
additional information
?
-
P0A9J4
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
?
-
Stenotrophomonas maltophilia 845
-
high specificity, among a variety of carbonyl compounds only ketopantoic acid and 2-keto-3-hydroxyisovalerate can serve as substrate
-
-
-
additional information
?
-
Escherichia coli BL21/lambdaDE3
-
-
-
-
-
additional information
?
-
Saccharomyces cerevisiae NRRL Y-2034
-
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
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
an essential step for the biosynthesis of pantothenate, i.e. vitamin B5
-
-
r
2-dehydropantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in the biosynthesis of pantothenate, i.e. vitamin B5
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
part of the pantothenate biosynthesis
-
-
?
2-oxopantoate + NADPH
(R)-pantoate + NADP+
show the reaction diagram
-
reaction is part of the D-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
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
-
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
thiamine synthesis, pantothenate and thiamine biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-, P0A9J4
pantothenate biosynthetic pathway
-
?
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
P0A9J4
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
-
the enzyme is involved in pantothenate, i.e. vitamin B5, biosynthesis, which is a precursor for CoA
-
-
r
ketopantoate + NADPH
pantoate + NADP+
show the reaction diagram
Escherichia coli BL21/lambdaDE3
-
-
-
?
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
alpha-NADPH
-
-
NADH
-
less active than NADPH at 0.1 mM
NADP+
-
cofactor binding structure and thermodynamics
NADP+
-
cofactor binding structure and thermodynamics, the cofactor is bound in the active site cleft between the N-terminal Rossmann-fold domain and the C-terminal alpha-helical domain
NADP+
-
protein-ligand interaction determination and analysis
NADP+
-
dependent on, binding structure of 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket, binding structure, overview
NADPH
-
B-specific hydrogen transfer
NADPH
-
cofactor binding structure and thermodynamics
NADPH
-
protein-ligand interaction determination and analysis
beta-NADPH
-
preference for beta-NADPH as cofactor
additional information
-
no activity with NADH
-
additional information
-
in presence of NADPH the ketopantoic acid reductase does not reduce artificial dye electron acceptors
-
additional information
-
-
-
additional information
-
NAD+/NADH are poor cofactors
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
Mg2+ independent
additional information
-
-
additional information
-
does not require Mg2+, independent of divalent metal ions
additional information
-
-
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
NaAsO2
-
carbonyl reagent
NADP+
-
competitive with NADPH at unsaturation level of ketopantoic acid
NADP+
-
competitive versus NADPH, noncompetitive versus ketopantoate
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
iodoacetate
-
sulfhydryl reagents, slightly inhibitory
additional information
-
ligand bindig modelling for inhibitor design
-
additional information
-
protein-ligand interaction determination and analysis
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
2-oxopantoate
-
pH 7.5, 25C, recombinant enzyme
3.4
-
2-oxopantoate
-
30C, pH 7.5
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
-
ketopantoate
-
+/-0.009, mutant K176C, alkylated
0.06
-
ketopantoate
P0A9J4
-
0.07
-
ketopantoate
-
+/-0.01, mutant K176C
0.12
-
ketopantoate
-
+/-0.008
0.12
-
ketopantoate
P0A9J4
-
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
0.007
-
NADP+
-
+/-0.002
0.002
-
NADPH
-
+/-0.0003, mutant E256A
0.0029
-
NADPH
-
+/-0.0006, mutant E256D
0.0038
-
NADPH
-
+/-0.0003, mutant K176C, alkylated
0.004
-
NADPH
-
+/-0.0004
0.004
-
NADPH
P0A9J4
-
0.0066
-
NADPH
-
+/-0.0016, mutant K176C
0.007
-
NADPH
-
pH 7.5, 27C, wild-type enzyme
0.016
-
NADPH
-
+/-0.003, mutant K176A
0.02
-
NADPH
P0A9J4
-
0.0318
-
NADPH
-
pH 7.0
0.26
-
Pantoate
-
+/-0.04
0.018
-
thio-NADPH
-
pH 7.5, 25C, recombinant enzyme
additional information
-
additional information
-
steady-state kinetics, pH-dependence of kinetics with different substrates, overview
-
additional information
-
additional information
-
kinetics and thermodynamics, wild-type enzyme, overview
-
additional information
-
additional information
-
kinetics and thermodynamics, overview
-
additional information
-
additional information
-
kinetics of recombinant wild-type and mutant enzymes, overview
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
25
-
2-dehydropantoate
-
pH 7.5, 27C, wild-type enzyme
8
-
ketopantoate
P0A9J4
-
40
-
ketopantoate
P0A9J4
-
25
-
NADPH
-
pH 7.5, 27C, wild-type enzyme
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
pH 7.6
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
-
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
-
-
-
additional information
-
-
no activity with pantoate, ketoisovalerate, oxaloacetate, pyruvate, 3-hydroxypyruvic acid, alpha-ketoglutarate, alpha-ketobutyrate, acetaldehyde at 0.5 mM
additional information
-
-
no activity for double mutant K176A/E256A
additional information
-
P0A9J4
-
additional information
-
-
3.8-fold higher specific activity with NADPH than with NADH
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
-
oxidation of D-pantoic acid
additional information
-
-
activity decreases with pH increase, reduction of ketopantoic acid
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
27
-
-
assay at
35
40
-
S-246, non-purified enzyme
35
40
-
AKU 309, non-purified enzyme
37
-
-
reduction of ketopantoic acid
42
-
-
reduction of ketopantoic acid at pH 6.0
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
15
60
-
15C: about 70% of activity maximum, 60C: about 85% of activity maximum
PDB
SCOP
CATH
ORGANISM
Bacillus subtilis (strain 168)
Cupriavidus pinatubonensis (strain JMP 134 / LMG 1197)
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)
Porphyromonas gingivalis (strain ATCC BAA-308 / W83)
Ralstonia solanacearum (strain Po82)
Staphylococcus aureus (strain Mu50 / ATCC 700699)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
33000
-
-
SDS-PAGE, gel filtration
33000
-
P0A9J4
SDS-PAGE
33800
-
-
calculated from 912 bp coding region
34000
-
-
gel filtration
34000
-
-
-
34000
-
P0A9J4
-
87000
-
-
+/-5000, sedimentation equilibrium method
115000
-
-
+/-5000, gel filtration
140000
160000
-
+/-5000, HPLC gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
monomer
-
1 * 33000 gel filtration, 1 * 33866 elelectrospray mass spectrometry analysis, 1 * 33870 amino acid sequence
monomer
P0A9J4
1 * 33000, gel filtration; 1 * 34000
monomer
Escherichia coli BL21/lambdaDE3
-
-
-
oligomer
-
3-5 * 30500 or 30000, SDS-PAGE
oligomer
Stenotrophomonas maltophilia 845
-
3-5 * 30500 or 30000, SDS-PAGE; 3-5 * 30500 or 30000, SDS-PAGE
-
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
side-chain modification
P0A9J4
SeMet substitution
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
prismic crystals, hanging drop vapor-diffusion technique
P0A9J4
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
-
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
-
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
-
fine needle, at about 40% ammonium sulfate saturation
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
stable below 60C for 10 min
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
-
-
30 min, pH 6-10
60
-
-
10 min, stable below 60C
70
-
-
10 min, 70% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
LITERATURE
anion-exchange chromatography, gel filtration chromatography
-
FPLC, anion exchange chromatography, gel filtration
P0A9J4
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
P0A9J4
gel chromatography
-
gel chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
gene ilvC, complementation of an enzyme-deficient Escherichia coli mutant strain
-
expression in Escherichia coli BL21(DE3)
-
expression in Escherichia coli K12
-
expression of wild-type and mutant enzymes
-
gene panE, expression of His6-tagged wild-type and mutant enzymes
-
KPR expressed using a pRSETA vector in Escherichia coli strain BL21(DE3)C41
P0A9J4
overexpression in strain BL21(DE3)
-
expression in Escherichia coli BL21/lambdaDE3
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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
E240A
-
site-directed mutagenesis, wild-type activity
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
E256D
-
wild-type activity
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
K176A/E256A
-
double mutant, no activity
K176C
-
wild-type activity
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
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
R31A
-
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
K72A
-
site-directed mutagenesis, altered steady-state kinetics of the mutant compared to the wild-type enzyme, 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
P0A9J4
Lys176 and Glu256 important for binding ketopantoate and the catalytic mechanism