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 | 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 | Comment | Organism | Structure |
---|---|---|---|
4-nitrophenol | inhibitory at concentrations above 1 mM | Burkholderia sp. |
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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Burkholderia sp. | Q2PWU9 | gene dntB | - |
Burkholderia sp. DNT | Q2PWU9 | gene dntB | - |
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 | 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 | Comment | Organism |
---|---|---|
4-methyl-5-nitrocatechol monooxygenase | - |
Burkholderia sp. |
4M5NC monooxygenase | - |
Burkholderia sp. |
dntB | - |
Burkholderia sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Burkholderia sp. |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Burkholderia sp. |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | enzyme DntB is a flavoprotein | Burkholderia sp. | |
NADPH | - |
Burkholderia sp. |
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 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. |