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Literature summary extracted from

  • Van Dillewijn, P.; Wittich, R.M.; Caballero, A.; Ramos, J.L.
    Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida (2008), Appl. Environ. Microbiol., 74, 6703-6708.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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

KM Value [mM]

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

Molecular Weight [Da]

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

Organism

EC Number Organism UniProt Comment Textmining
1.7.1.B1 Pseudomonas putida Q88PD0
-
-
1.7.1.B1 Pseudomonas putida KT 2240 Q88PD0
-
-

Purification (Commentary)

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

Reaction

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
a
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
a

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

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 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.7.1.B1 monomer in solution Pseudomonas putida

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.7.1.B1 25
-
 assay at Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.7.1.B1 7
-
 assay at Pseudomonas putida

Cofactor

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