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Literature summary for 1.14.13.210 extracted from

  • Leungsakul, T.; Johnson, G.R.; Wood, T.K.
    Protein engineering of the 4-methyl-5-nitrocatechol monooxygenase from Burkholderia sp. strain DNT for enhanced degradation of nitroaromatics (2006), Appl. Environ. Microbiol., 72, 3933-3939.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.14.13.210

Cloned(Commentary)

Cloned (Comment) 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.

Protein Variants

Protein Variants Comment 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 Comment Organism Structure
4-nitrophenol inhibitory at concentrations above 1 mM Burkholderia sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment 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 Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4-methyl-5-nitrocatechol + O2 + NADPH + H+ Burkholderia sp.
-
 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?
4-methyl-5-nitrocatechol + O2 + NADPH + H+ Burkholderia sp. DNT
-
 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?

Organism

Organism UniProt Comment Textmining
Burkholderia sp. Q2PWU9 gene dntB
-
Burkholderia sp. DNT Q2PWU9 gene dntB
-

Purification (Commentary)

Purification (Comment) 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-methyl-4-nitrophenol + O2 + NADPH + H+ low activity with the wild-type enzyme, higher activity with enzyme mutant M22L/L380I 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 Burkholderia sp. DNT 2-methyl-4-benzoquinone + nitrite + H2O + NADP+
-
 ?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?
4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
 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 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 Burkholderia sp. DNT 4-benzoquinone + nitrite + H2O + NADP+
-
 ?
5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. 2-hydroxyquinone + nitrite + H2O + NADP+
-
 ?
5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. DNT 2-hydroxyquinone + nitrite + H2O + NADP+
-
 ?
additional information the enzyme DntB has a very narrow substrate range Burkholderia sp. ?
-
 ?
additional information the enzyme DntB has a very narrow substrate range Burkholderia sp. DNT ?
-
 ?

Synonyms

Synonyms Comment Organism
4-methyl-5-nitrocatechol monooxygenase
-
 Burkholderia sp.
4M5NC monooxygenase
-
 Burkholderia sp.
dntB
-
 Burkholderia sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Burkholderia sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment 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 Comment Organism
8
-
 assay at Burkholderia sp.

Cofactor

Cofactor Comment Organism Structure
FAD enzyme DntB is a flavoprotein Burkholderia sp.
NADPH
-
 Burkholderia sp.

General Information

General Information Comment 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 Comment 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.