EC Number | Cloned (Comment) | Organism |
---|---|---|
1.7.1.B1 | xenB gene is amplified using appropriate primers with BamHI and HindIII sites and Pseudomonas putida KT2440 chromosomal DNA as a template. After digestion with restriction enzymes, the PCR product is ligated into the pET28b(+) vector. Resulting plasmid contains the coding sequence in frame with a DNA sequence encoding a His-tag, which resulted in a hexahistidine tail. For protein-His6 expression, plasmid is transformed into Escherichia coli BL21. | Pseudomonas putida |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.7.1.B1 | 0.027 | - |
glycerol trinitrate | commentary | Pseudomonas putida | |
1.7.1.B1 | 0.037 | - |
2,4,6-trinitrotoluene | - |
Pseudomonas putida | |
1.7.1.B1 | 0.044 | - |
N-ethylmaleimide | commentary | Pseudomonas putida | |
1.7.1.B1 | 0.077 | - |
NADPH | - |
Pseudomonas putida | |
1.7.1.B1 | 0.158 | - |
cyclohexenone | commentary | Pseudomonas putida |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
1.7.1.B1 | additional information | - |
determined by gel filtration, molecular masses of the His6-tagged proteins XenA to XenF are in the range of 40900 to 42600 Da | Pseudomonas putida |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.7.1.B1 | Pseudomonas putida | Q88PD0 | - |
- |
1.7.1.B1 | Pseudomonas putida KT 2240 | Q88PD0 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
1.7.1.B1 | His6-tagged protein is purified by nickel affinity chromatography and eluted with a continuous imidazole gradient. | Pseudomonas putida |
EC Number | Reaction | Comment | Organism | Reaction ID |
---|---|---|---|---|
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In adidtion the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite | Pseudomonas putida | |
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite | Pseudomonas putida |
EC Number | Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|---|
1.7.1.B1 | additional information | - |
Vmax is denoted with 112 micromol/min/mg protein, XenB exhibits the highest Vmax values and the most favorable Vmax/Km relationship for 2,4,6-trinitrotoluene compared to those of the other active xenobiotic reductases of Pseudomonas putida KT2440 | Pseudomonas putida |
1.7.1.B1 | 1.88 | - |
highest specific activity within xenobiotic reductases A to F | Pseudomonas putida |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ | - |
Pseudomonas putida | N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite | ? | |
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ | - |
Pseudomonas putida KT 2240 | N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite | ? | |
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ | - |
Pseudomonas putida | N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite | ? | |
1.7.1.B1 | 2,4,6-trinitrotoluene + 2 NADPH + 2 H+ | - |
Pseudomonas putida KT 2240 | N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O | i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite | ? | |
1.7.1.B1 | cyclohexenone + NADPH + H+ | - |
Pseudomonas putida | ? | - |
? | |
1.7.1.B1 | cyclohexenone + NADPH + H+ | - |
Pseudomonas putida KT 2240 | ? | - |
? | |
1.7.1.B1 | glycerol trinitrate + NADPH + H+ | - |
Pseudomonas putida | ? | - |
? | |
1.7.1.B1 | glycerol trinitrate + NADPH + H+ | - |
Pseudomonas putida KT 2240 | ? | - |
? | |
1.7.1.B1 | additional information | ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo | Pseudomonas putida | ? | - |
? | |
1.7.1.B1 | additional information | ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo | Pseudomonas putida KT 2240 | ? | - |
? | |
1.7.1.B1 | N-ethylmaleimide + NADPH + H+ | - |
Pseudomonas putida | ? | - |
? |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
1.7.1.B1 | monomer | in solution | Pseudomonas putida |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.7.1.B1 | old yellow enzyme | - |
Pseudomonas putida |
1.7.1.B1 | OYE | - |
Pseudomonas putida |
1.7.1.B1 | XenB | - |
Pseudomonas putida |
1.7.1.B1 | xenobiotic reductase B | family of flavoproteins | Pseudomonas putida |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.7.1.B1 | 25 | - |
assay at | Pseudomonas putida |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.7.1.B1 | 7 | - |
assay at | Pseudomonas putida |
EC Number | Cofactor | Comment | Organism | Structure |
---|---|---|---|---|
1.7.1.B1 | FMN | in the primary sequence of these proteins, a number of residues involved in interactions with the FMN cofactor is found | Pseudomonas putida | |
1.7.1.B1 | NAD(P)H | enzyme preferentially uses NADPH as a cofactor | Pseudomonas putida |