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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.210 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

EC Number Protein Variants Comment Organism
1.14.13.210 L380I site-directed mutagenesis, the mutant shows unaltered activity with 4-nitrophenol compared to the wild-type enzyme Burkholderia sp.
1.14.13.210 M22L site-directed mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme Burkholderia sp.
1.14.13.210 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.
1.14.13.210 M22L/L380M simultaneous saturation mutagenesis, the mutant shows slightly reduced activity with 4-nitrophenol compared to the wild-type enzyme Burkholderia sp.
1.14.13.210 M22S/L380V simultaneous saturation mutagenesis, the mutant shows 20% enhanced degradation of 4-nitrophenol compared to mutant M22L/L380I Burkholderia sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.13.210 4-nitrophenol inhibitory at concentrations above 1 mM Burkholderia sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.210 additional information
-
 additional information Michaelis-Menten kinetics Burkholderia sp.
1.14.13.210 0.1
-
 4-nitrophenol mutant M22L/L380I, pH 8.0, 37°C Burkholderia sp.
1.14.13.210 0.35
-
 4-nitrophenol wild-type enzyme, pH 8.0, 37°C Burkholderia sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.210 4-methyl-5-nitrocatechol + O2 + NADPH + H+ Burkholderia sp.
-
 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?
1.14.13.210 4-methyl-5-nitrocatechol + O2 + NADPH + H+ Burkholderia sp. DNT
-
 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.210 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.210 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+
-
 ?
1.14.13.210 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+
-
 ?
1.14.13.210 4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?
1.14.13.210 4-methyl-5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. DNT 2-hydroxy-5-methylquinone + nitrite + H2O + NADP+
-
 ?
1.14.13.210 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+
-
 ?
1.14.13.210 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+
-
 ?
1.14.13.210 5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. 2-hydroxyquinone + nitrite + H2O + NADP+
-
 ?
1.14.13.210 5-nitrocatechol + O2 + NADPH + H+
-
 Burkholderia sp. DNT 2-hydroxyquinone + nitrite + H2O + NADP+
-
 ?
1.14.13.210 additional information the enzyme DntB has a very narrow substrate range Burkholderia sp. ?
-
 ?
1.14.13.210 additional information the enzyme DntB has a very narrow substrate range Burkholderia sp. DNT ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.13.210 4-methyl-5-nitrocatechol monooxygenase
-
 Burkholderia sp.
1.14.13.210 4M5NC monooxygenase
-
 Burkholderia sp.
1.14.13.210 dntB
-
 Burkholderia sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.210 37
-
 assay at Burkholderia sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.13.210 0.014
-
 4-nitrophenol wild-type enzyme, pH 8.0, 37°C Burkholderia sp.
1.14.13.210 0.015
-
 4-nitrophenol mutant M22L/L380I, pH 8.0, 37°C Burkholderia sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.210 8
-
 assay at Burkholderia sp.

Cofactor

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

General Information

EC Number General Information Comment Organism
1.14.13.210 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.210 0.04
-
 4-nitrophenol wild-type enzyme, pH 8.0, 37°C Burkholderia sp.
1.14.13.210 0.45
-
 4-nitrophenol mutant M22L/L380I, pH 8.0, 37°C Burkholderia sp.