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show all sequences of 1.14.13.210

Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics

Leungsakul, T.; Johnson, G.R.; Wood, T.K.; Appl. Environ. Microbiol. 72, 3933-3939 (2006)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene dntB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TG1
Burkholderia sp.
Engineering
Amino acid exchange
Commentary
Organism
L380I
site-directed mutagenesis, the mutant shows unaltered activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22L
site-directed mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22L/L380I
error-prone PCR, protein engineering for nitrite removal, the mutant enzyme accepts two addtional substrates 4-nitrophenol and 3-methyl-4-nitrophenol. With 4-nitrophenol, the initial rate of the mutant M22L/L380I enzyme is 10fold higher than that of the wild-type enzyme, the catalytic efficiency for 4-nitrophenol degradation is 11fold higher, and with 3-methyl-4-nitrophenol, the initial rate is 4fold higher compared to the wild-type enzyme
Burkholderia sp.
M22L/L380M
simultaneous saturation mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22S/L380V
simultaneous saturation mutagenesis, the mutant shows 20% enhanced degradation of 4-nitrophenol compared to mutant M22L/L380I
Burkholderia sp.
Inhibitors
Inhibitors
Commentary
Organism
Structure
4-nitrophenol
inhibitory at concentrations above 1 mM
Burkholderia sp.
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Burkholderia sp.
0.1
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
0.35
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4-methyl-5-nitrocatechol + O2 + NADPH + H+
Burkholderia sp.
-
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
Burkholderia sp. DNT
-
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Burkholderia sp.
Q2PWU9
gene dntB
-
Burkholderia sp. DNT
Q2PWU9
gene dntB
-
Purification (Commentary)
Commentary
Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
Burkholderia sp.
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-methyl-4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp.
2-methyl-4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
3-methyl-4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp. DNT
2-methyl-4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp. DNT
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp. DNT
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
-
-
?
4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp.
4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp. DNT
4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxyquinone + nitrite + H2O + NADP+
-
-
-
?
additional information
the enzyme DntB has a very narrow substrate range
735475
Burkholderia sp.
?
-
-
-
-
additional information
the enzyme DntB has a very narrow substrate range
735475
Burkholderia sp. DNT
?
-
-
-
-
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Burkholderia sp.
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.014
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
0.015
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Burkholderia sp.
Cofactor
Cofactor
Commentary
Organism
Structure
FAD
enzyme DntB is a flavoprotein
Burkholderia sp.
NADPH
-
Burkholderia sp.
Cloned(Commentary) (protein specific)
Commentary
Organism
gene dntB, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain TG1
Burkholderia sp.
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FAD
enzyme DntB is a flavoprotein
Burkholderia sp.
NADPH
-
Burkholderia sp.
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
L380I
site-directed mutagenesis, the mutant shows unaltered activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22L
site-directed mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22L/L380I
error-prone PCR, protein engineering for nitrite removal, the mutant enzyme accepts two addtional substrates 4-nitrophenol and 3-methyl-4-nitrophenol. With 4-nitrophenol, the initial rate of the mutant M22L/L380I enzyme is 10fold higher than that of the wild-type enzyme, the catalytic efficiency for 4-nitrophenol degradation is 11fold higher, and with 3-methyl-4-nitrophenol, the initial rate is 4fold higher compared to the wild-type enzyme
Burkholderia sp.
M22L/L380M
simultaneous saturation mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme
Burkholderia sp.
M22S/L380V
simultaneous saturation mutagenesis, the mutant shows 20% enhanced degradation of 4-nitrophenol compared to mutant M22L/L380I
Burkholderia sp.
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
4-nitrophenol
inhibitory at concentrations above 1 mM
Burkholderia sp.
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Burkholderia sp.
0.1
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
0.35
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4-methyl-5-nitrocatechol + O2 + NADPH + H+
Burkholderia sp.
-
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
Burkholderia sp. DNT
-
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis
Burkholderia sp.
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-methyl-4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp.
2-methyl-4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
3-methyl-4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp. DNT
2-methyl-4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp. DNT
2-hydroxy-5-methylquinone + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
-
-
?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp. DNT
2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
-
-
?
4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp.
4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
4-nitrophenol + O2 + NADPH + H+
low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I
735475
Burkholderia sp. DNT
4-benzoquinone + nitrite + H2O + NADP+
-
-
-
?
5-nitrocatechol + O2 + NADPH + H+
-
735475
Burkholderia sp.
2-hydroxyquinone + nitrite + H2O + NADP+
-
-
-
?
additional information
the enzyme DntB has a very narrow substrate range
735475
Burkholderia sp.
?
-
-
-
-
additional information
the enzyme DntB has a very narrow substrate range
735475
Burkholderia sp. DNT
?
-
-
-
-
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Burkholderia sp.
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.014
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
0.015
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Burkholderia sp.
General Information
General Information
Commentary
Organism
metabolism
4-methyl-5-nitrocatechol monooxygenase catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4-methyl-5-nitrocatechol to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group
Burkholderia sp.
General Information (protein specific)
General Information
Commentary
Organism
metabolism
4-methyl-5-nitrocatechol monooxygenase catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4-methyl-5-nitrocatechol to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group
Burkholderia sp.
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.04
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
0.45
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.04
-
4-nitrophenol
wild-type enzyme, pH 8.0, 37°C
Burkholderia sp.
0.45
-
4-nitrophenol
mutant M22L/L380I, pH 8.0, 37°C
Burkholderia sp.
Other publictions for EC 1.14.13.210
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)
735475
Leungsakul
Protein engineering of the 4-m ...
Burkholderia sp., Burkholderia sp. DNT
Appl. Environ. Microbiol.
72
3933-3939
2006
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1
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5
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1
3
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11
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11
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1
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1
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2
2
736341
Haigler
Purification and sequence anal ...
Burkholderia sp., Burkholderia sp. RASC
J. Bacteriol.
178
6019-6024
1996
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1
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3
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1
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6
1
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3
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1
3
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1
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6
1
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1
1
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736336
Haigler
Biodegradation of 4-methyl-5-n ...
Pseudomonas sp., Pseudomonas sp. DNT
J. Bacteriol.
176
3433-3437
1994
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