BRENDA - Enzyme Database show
show all sequences of 1.1.1.169

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue

Zheng, R.; Blanchard, J.S.; Biochemistry 39, 16244-16251 (2000)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
bromoethylamine
activation of enzyme from K176C mutant
Escherichia coli
ethylamine
activation of enzyme from K176A mutant
Escherichia coli
formate
E256A mutant activity greatly increased, twice as high at pH 7.2 as at pH 5.9
Escherichia coli
methylamine
activation of enzyme from K176A mutant
Escherichia coli
Propylamine
activation of enzyme from K176A mutant
Escherichia coli
Cloned(Commentary)
Commentary
Organism
expression in Escherichia coli BL21(DE3)
Escherichia coli
Engineering
Amino acid exchange
Commentary
Organism
D248A
site-directed mutagenesis, wild-type activity
Escherichia coli
E210A
site-directed mutagenesis, wild-type activity
Escherichia coli
E240A
site-directed mutagenesis, wild-type activity
Escherichia coli
E256A
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
Escherichia coli
E256D
wild-type activity
Escherichia coli
K176A
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
Escherichia coli
K176A/E256A
double mutant, no activity
Escherichia coli
K176C
wild-type activity
Escherichia coli
K72A
site-directed mutagenesis, wild-type activity
Escherichia coli
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
Escherichia coli
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.002
-
NADPH
+/-0.0003, mutant E256A
Escherichia coli
0.0029
-
NADPH
+/-0.0006, mutant E256D
Escherichia coli
0.0038
-
NADPH
+/-0.0003, mutant K176C, alkylated
Escherichia coli
0.004
-
NADPH
+/-0.0004
Escherichia coli
0.0066
-
NADPH
+/-0.0016, mutant K176C
Escherichia coli
0.016
-
NADPH
+/-0.003, mutant K176A
Escherichia coli
0.038
-
ketopantoate
+/-0.009, mutant K176C, alkylated
Escherichia coli
0.07
-
ketopantoate
+/-0.01, mutant K176C
Escherichia coli
0.12
-
ketopantoate
+/-0.008
Escherichia coli
0.95
-
ketopantoate
+/-0.29, mutant E256D
Escherichia coli
7.5
-
ketopantoate
+/-2.9, mutant K256A
Escherichia coli
40
-
ketopantoate
+/-6, mutant K176A
Escherichia coli
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
additional information
-
Escherichia coli
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
34000
-
-
Escherichia coli
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Escherichia coli
-
expressed in BL21(DE3)
-
Purification (Commentary)
Commentary
Organism
homogeneity in a yield of 20-60 mg from 3-6 g of cells
Escherichia coli
Specific Activity [micromol/min/mg]
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
additional information
-
no activity for double mutant K176A/E256A
Escherichia coli
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
alpha-ketopantoate + NADPH
-
286109
Escherichia coli
D-pantoate + NADP+
-
286109
Escherichia coli
r
additional information
no activity for double mutant K176A/E256A
286109
Escherichia coli
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
monomer
-
Escherichia coli
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
additional information
-
activity decreases with pH increase, reduction of ketopantoic acid
Escherichia coli
Cofactor
Cofactor
Commentary
Organism
Structure
NADP+
-
Escherichia coli
NADPH
-
Escherichia coli
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
bromoethylamine
activation of enzyme from K176C mutant
Escherichia coli
ethylamine
activation of enzyme from K176A mutant
Escherichia coli
formate
E256A mutant activity greatly increased, twice as high at pH 7.2 as at pH 5.9
Escherichia coli
methylamine
activation of enzyme from K176A mutant
Escherichia coli
Propylamine
activation of enzyme from K176A mutant
Escherichia coli
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli BL21(DE3)
Escherichia coli
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADP+
-
Escherichia coli
NADPH
-
Escherichia coli
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D248A
site-directed mutagenesis, wild-type activity
Escherichia coli
E210A
site-directed mutagenesis, wild-type activity
Escherichia coli
E240A
site-directed mutagenesis, wild-type activity
Escherichia coli
E256A
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
Escherichia coli
E256D
wild-type activity
Escherichia coli
K176A
site-directed mutagenesis, significant reduction in catalytic efficiency of enzyme
Escherichia coli
K176A/E256A
double mutant, no activity
Escherichia coli
K176C
wild-type activity
Escherichia coli
K72A
site-directed mutagenesis, wild-type activity
Escherichia coli
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
Escherichia coli
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.002
-
NADPH
+/-0.0003, mutant E256A
Escherichia coli
0.0029
-
NADPH
+/-0.0006, mutant E256D
Escherichia coli
0.0038
-
NADPH
+/-0.0003, mutant K176C, alkylated
Escherichia coli
0.004
-
NADPH
+/-0.0004
Escherichia coli
0.0066
-
NADPH
+/-0.0016, mutant K176C
Escherichia coli
0.016
-
NADPH
+/-0.003, mutant K176A
Escherichia coli
0.038
-
ketopantoate
+/-0.009, mutant K176C, alkylated
Escherichia coli
0.07
-
ketopantoate
+/-0.01, mutant K176C
Escherichia coli
0.12
-
ketopantoate
+/-0.008
Escherichia coli
0.95
-
ketopantoate
+/-0.29, mutant E256D
Escherichia coli
7.5
-
ketopantoate
+/-2.9, mutant K256A
Escherichia coli
40
-
ketopantoate
+/-6, mutant K176A
Escherichia coli
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
additional information
-
Escherichia coli
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
34000
-
-
Escherichia coli
Purification (Commentary) (protein specific)
Commentary
Organism
homogeneity in a yield of 20-60 mg from 3-6 g of cells
Escherichia coli
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
additional information
-
no activity for double mutant K176A/E256A
Escherichia coli
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
alpha-ketopantoate + NADPH
-
286109
Escherichia coli
D-pantoate + NADP+
-
286109
Escherichia coli
r
additional information
no activity for double mutant K176A/E256A
286109
Escherichia coli
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
monomer
-
Escherichia coli
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
additional information
-
activity decreases with pH increase, reduction of ketopantoic acid
Escherichia coli
Other publictions for EC 1.1.1.169
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [░C]
Temperature Range [░C]
Temperature Stability [░C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [░C] (protein specific)
Temperature Range [░C] (protein specific)
Temperature Stability [░C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
725293
Miller
PanG, a new ketopantoate reduc ...
Francisella tularensis subsp. novicida
J. Bacteriol.
195
965-976
2013
-
-
-
-
-
-
-
-
-
-
-
-
-
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
736827
Tomita
Identification and characteriz ...
Thermococcus kodakarensis, Thermococcus kodakarensis KUW1
Mol. Microbiol.
90
307-321
2013
-
-
1
-
1
-
1
7
-
-
2
4
-
8
-
-
1
-
-
-
-
-
13
1
1
1
1
6
1
-
-
4
1
-
-
-
-
1
4
-
1
-
-
1
1
7
-
-
2
4
-
-
-
1
-
-
-
-
13
1
1
1
1
6
1
-
-
-
-
2
2
-
6
6
696603
Headey
Backbone assignments of the 34 ...
Escherichia coli
Biomol. NMR Assign.
2
93-96
2008
-
-
1
-
-
-
-
-
-
-
1
-
-
1
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
684140
Ciulli
pH-tuneable binding of 2-phosp ...
Escherichia coli
Acta Crystallogr. Sect. D
63
171-178
2007
-
-
-
1
-
-
-
-
-
-
-
1
-
2
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
687675
Ciulli
Crystal structure of Escherich ...
Escherichia coli
J. Biol. Chem.
282
8487-8497
2007
-
-
1
1
6
-
-
3
-
-
-
1
-
2
-
-
-
-
-
-
-
-
2
-
1
-
-
2
1
-
-
2
-
-
-
-
-
1
2
1
6
-
-
-
-
3
-
-
-
1
-
-
-
-
-
-
-
-
2
-
1
-
-
2
1
-
-
-
-
-
-
-
-
-
669806
Ciulli
Probing hot spots at protein-l ...
Escherichia coli
J. Med. Chem.
49
4992-5000
2006
-
-
-
-
-
-
7
1
-
-
-
1
-
2
-
-
-
1
-
-
-
-
2
-
-
-
-
-
1
-
-
3
6
-
-
-
-
-
3
-
-
-
-
7
6
1
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
667561
Ciulli
Biophysical tools to monitor e ...
Escherichia coli
Biochem. Soc. Trans.
33
767-771
2005
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
667687
Lobley
The crystal structure of Esche ...
Escherichia coli
Biochemistry
44
8930-8939
2005
-
-
1
1
7
-
-
1
-
-
-
1
-
2
-
-
1
1
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
1
7
-
-
-
-
1
-
-
-
1
-
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
654677
Zheng
Substrate specificity and kine ...
Escherichia coli
Biochemistry
42
11289-11296
2003
-
-
1
-
-
-
-
8
-
-
-
1
-
2
-
-
-
-
-
-
-
-
7
-
1
-
-
-
1
1
-
4
-
-
-
-
-
1
4
-
-
-
-
-
-
8
-
-
-
1
-
-
-
-
-
-
-
-
7
-
1
-
-
-
1
1
-
-
-
-
-
-
-
-
656330
Merkamm
Ketopantoate reductase activit ...
Corynebacterium glutamicum
J. Biotechnol.
104
253-260
2003
-
-
1
-
-
-
-
1
-
-
-
1
-
3
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
-
-
2
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
286110
Matak-Vinkovic
Crystal structure of Escherich ...
Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, Stenotrophomonas maltophilia, Stenotrophomonas maltophilia 845
Biochemistry
40
14493-14500
2001
-
-
1
1
1
-
-
4
-
2
4
2
-
7
-
1
1
-
-
-
2
-
4
3
-
-
-
2
-
-
-
3
-
-
-
-
-
1
3
1
1
-
-
-
-
4
-
2
4
2
-
-
1
1
-
-
2
-
4
3
-
-
-
2
-
-
-
-
-
-
-
-
-
-
286108
Zheng
Kinetic and mechanistic analys ...
Escherichia coli
Biochemistry
39
3708-3717
2000
-
-
1
-
-
-
2
4
-
1
4
1
-
2
-
-
1
-
-
-
1
-
2
1
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
-
-
-
-
2
-
4
-
1
4
1
-
-
-
1
-
-
1
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
286109
Zheng
Identification of active site ...
Escherichia coli
Biochemistry
39
16244-16251
2000
5
-
1
-
10
-
-
12
-
1
1
-
-
2
-
-
1
-
-
-
1
-
2
1
-
-
-
-
1
-
-
2
-
-
-
5
-
1
2
-
10
-
-
-
-
12
-
1
1
-
-
-
-
1
-
-
1
-
2
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
286106
Elischewski
Pantothenate production in Esc ...
Escherichia coli
J. Biotechnol.
75
135-146
1999
-
-
1
-
-
-
-
-
-
-
1
-
-
3
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
286107
Frodyma
ApbA, the ketopantoate reducta ...
Escherichia coli, Escherichia coli BL21/lambdaDE3, Saccharomyces cerevisiae, Salmonella enterica subsp. enterica serovar Typhimurium, Stenotrophomonas maltophilia 845, Stenotrophomonas maltophilia
J. Biol. Chem.
273
5572-5576
1998
-
-
1
-
-
-
-
2
-
-
1
3
-
10
-
-
1
-
-
-
6
-
12
2
1
-
-
-
1
-
-
8
-
-
-
-
-
1
8
-
-
-
-
-
-
2
-
-
1
3
-
-
-
1
-
-
6
-
12
2
1
-
-
-
1
-
-
-
-
-
-
-
-
-
286102
Kataoka
-
Novel enzymic production of D- ...
Agrobacterium sp., Agrobacterium tumefaciens, Pseudomonas putida, Stenotrophomonas maltophilia
Agric. Biol. Chem.
54
177-182
1990
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
4
-
2
-
-
-
-
-
-
3
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
286103
Shimizu
Ketopantoic acid reductase of ...
Saccharomyces cerevisiae, Stenotrophomonas maltophilia, Stenotrophomonas maltophilia 845
J. Biol. Chem.
263
12077-12084
1988
-
-
-
1
-
-
10
4
-
1
4
-
-
6
-
-
1
-
-
-
2
1
6
1
1
1
3
-
2
-
2
4
-
-
-
-
-
-
4
1
-
-
-
10
-
4
-
1
4
-
-
-
-
1
-
-
2
1
6
1
1
1
3
-
2
-
2
-
-
-
-
-
-
-
286105
Wilken
Ketopantoic acid and ketopanto ...
Escherichia coli, Saccharomyces cerevisiae
J. Biol. Chem.
250
2311-2314
1975
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
2
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
286104
King
-
Separation and preliminary stu ...
Saccharomyces cerevisiae, Saccharomyces cerevisiae NRRL Y-2034
J. Biol. Chem.
247
4096-4098
1972
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
1
-
-
-
2
-
4
-
-
-
-
-
2
1
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
2
-
4
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-