Information on EC 1.7.1.B1 - xenobiotic reductase B

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The expected taxonomic range for this enzyme is: Pseudomonas

EC NUMBER
COMMENTARY hide
1.7.1.B1
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
xenobiotic reductase B
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
show the reaction diagram
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
reduction
SYSTEMATIC NAME
IUBMB Comments
2,4,6-trinitrotoluene:NADPH oxidoreductase
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
show the reaction diagram
2,4,6-trinitrotoluene + 2 NADPH + 2 H+
N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O
show the reaction diagram
cyclohexenone + NADPH + H+
?
show the reaction diagram
glycerol trinitrate + NADPH + H+
?
show the reaction diagram
N-ethylmaleimide + NADPH + H+
?
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
FMN
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in the primary sequence of these proteins, a number of residues involved in interactions with the FMN cofactor is found
NAD(P)H
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enzyme preferentially uses NADPH as a cofactor
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NH4Cl
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included in assay medium
Zn2+
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included in assay medium
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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Ag+
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about 70% inhibition
Cu2+
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about 70% inhibition
Hg2+
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about 70% inhibition
additional information
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not inhibitory: Fe2+
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.037 - 0.048
2,4,6-trinitrotoluene
0.158
cyclohexenone
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commentary
0.027
glycerol trinitrate
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commentary
0.044
N-ethylmaleimide
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commentary
0.013 - 0.077
NADPH
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.88
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highest specific activity within xenobiotic reductases A to F
5.01
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pH 7.0, 30°C
105
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pH 7.0
additional information
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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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
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x * 27000, SDS-PAGE
additional information
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determined by gel filtration, molecular masses of the His6-tagged proteins XenA to XenF are in the range of 40900 to 42600 Da
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 27000, SDS-PAGE
monomer
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel affinity chromatography and eluted with a continuous imidazole gradient
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by nickel affinity chromatography using a continuous imidazole gradient
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His6-tagged protein is purified by nickel affinity chromatography and eluted with a continuous imidazole gradient.
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
For His-tagged protein production, plasmid is transformed into Escherichia coli BL21 pLysS.
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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.
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression of enzyme gene xenB is induced by S-nitrosoglutathione in a mutant lacking multidrug-resistance system MexEF-OprN
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presence of chloramphenicol does not induce expression of enzyme gene
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