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Enzyme Nomenclature
Information on EC 1.7.1.B3 - aromatic nitroreductase [NADPH]
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
1.7.1.B3
preliminary BRENDA-supplied EC number
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RECOMMENDED NAME
GeneOntology No.
aromatic nitroreductase [NADPH]
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
an aromatic amine + NADP+ + H2O = an aromatic hydroxylamine + NADPH + H+
(1a)
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an aromatic hydroxylamine + NADP+ = an aromatic nitric oxide + NADPH + H+
(1b)
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an aromatic nitric oxide + NADP+ + H2O = an aromatic nitrate + NADPH + H+
(1c)
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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
in vivo the reaction is obseverved in the opposite direction from that shown in the equation; (overall reaction)
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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
catalytic mechanism analysis, two (four)-electron reduction of nitrobenzenes and single-step (H-) hydride transfer mechanism. NfsA follows a ping-pong mechanism, overview
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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism
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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
the enzyme uses NADPH as the hydrogen donor to catalyze the reduction of a nitro group by a ping-pong Bi-Bi kinetic mechanism
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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism
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SYSTEMATIC NAME
IUBMB Comments
aromatic nitrate:NADPH oxidroreductase
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
additional information
evolution
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analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview
evolution
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nitroreductases are a family of flavoproteins that catalyze the NAD(P)H-dependent reduction of nitro groups to hydroxylamino and/or amino groups on various nitro-substituted compounds
evolution
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analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview
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physiological function
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NfsA has potential applications in the biodegradation of nitroaromatic environment pollutants, e.g. explosives, and is also of interest for the anticancer strategy gene-directed enzyme prodrug therapy
physiological function
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residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA, Phe42 is important for maintaining NfsA activity and structure
additional information
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comparisons of the quantitative structure-activity relationships of single-electron reduction of quinones and nitroaromatic compounds by dehydrogenase (electron transferase) flavoenzymes, overview
additional information
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the enzyme crystal structure of NfsA, PDB ID 1F5V, is used for molecular ligand docking studies
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
nitro-CBI-5-[(dimethylamino)ethoxy]indole + NAD(P)H + H+
? + NAD(P)+
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a nitro-CBI prodrug, low activity
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-
?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
2,4,6-trinitrotoluene + NADPH + H+
4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
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-
-
-
?
2,4,6-trinitrotoluene + NADPH + H+
4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
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i.e. TNT, NfsA reduces TNT to the para isomer 4-hydroxylamino-2,6-dinitrotoluene more easily than the ortho isomer 2-hydroxylamino-4,6-dinitrotoluene
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-
?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+
? + NADP+
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i.e. CB1954, an aziridinyl dinitrobenzamide prodrug
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-
?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+
? + NADP+
i.e. CB1954, an aziridinyl dinitrobenzamide prodrug
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?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+
? + NADP+
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i.e. CB1954, an aziridinyl dinitrobenzamide prodrug
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-
?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
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-
?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
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?
additional information
?
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the enzyme reduces both nitrofurazone and FMN effectively, see for EC 1.5.1.38, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity
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additional information
?
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an intrinsically oxygen-insensitive nature of the two-electron reduction mechanism
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additional information
?
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NADPH-dependent reduction of quinones, cf. EC 1.6.5.5, and nitroaromatic compounds by NfsA, overview. The reactivity of nitroaromatic compounds (log kcat/Km) follows a linear dependence on their single-electron reduction potential, indicating a limited role for compound structure or active site flexibility in their reactivity. The reactivity of quinones is lower than that of nitroaromatics having similar single-electron reduction potential values, except for the significantly enhanced reactivity of 2-OH-1,4-naphthoquinones. The reduction of quinones by NfsA is most consistent with a single-step (H-) hydride transfer mechanism, quantitative analysis of two-electron reduction of quinones and nitroaromatics, overview
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additional information
?
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NfsA has a broad substrate spectrum. Various nitroaromatic substrates, including 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), 2,4,6-trinitrotoluene (TNT), and 2,4-dinitrotoluene (2,4-DNT), can serve as substrates
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additional information
?
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no activity with 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2,4,6-trinitrotoluene + NADPH + H+
4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
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?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
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?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FMN
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tightly associated to, two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
dicoumarol
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inhibits Escherichia coli enzyme NfsB far more strongly than enzyme NfsA, by acting as a competitive inhibitor towards NADPH, uncompetitive towards substrate tetryl
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.3257
2-amino-4,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
0.6427
4-amino-2,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
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0.322
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
0.327
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant wild-type enzyme, pH 7.0, 22°C
0.332
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
0.487
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
0.085
NADPH
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recombinant mutant F46Y, pH 7.0, 22°C; recombinant wild-type enzyme, pH 7.0, 22°C
additional information
additional information
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Michaelis-Menten steady-state kinetics. For NfsA the oxidative half-reaction (i.e., the reoxidation of FMNH by the oxidant substrate) is a rate-limiting step, because the values of kcat at infinite concentrations of tetryl or 2,4,6-trinitrotoluene are substantially lower than the lowest rate of the reductive half reaction (the reduction of FMN by NADPH) measured in the preliminary rapid reaction experiments. Stopped-flow and single-turnover measurements
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additional information
additional information
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enzyme kinetic analysis, overview
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additional information
additional information
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Michaelis-Menten steady-state kinetics
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additional information
additional information
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Michaelis-Menten steady-state kinetics of recombinant His6-tagged enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
12.36
2-amino-4,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
1.05
4-amino-2,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
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1.08
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
2.99
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
24.27
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
36.64
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant wild-type enzyme, pH 7.0, 22°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
38
2-amino-4,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
1.6
4-amino-2,6-dinitrotoluene
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pH 7.0, 37°C, recombinant His6-tagged enzyme
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2
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
9
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
75
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant mutant F46Y, pH 7.0, 22°C
112
5-(aziridin-1-yl)-2,4-dinitrobenzamide
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recombinant wild-type enzyme, pH 7.0, 22°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.1
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purified recombinant mutant F46A, pH 7.0, 22°C, substrate nitrofurazone
0.29
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purified recombinant mutant F46N, pH 7.0, 22°C, substrate nitrofurazone
3.74
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purified recombinant mutant F46Y, pH 7.0, 22°C, substrate nitrofurazone
7.75
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purified recombinant wild-type enzyme, pH 7.0, 22°C, substrate nitrofurazone
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
homodimer
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2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
homodimer
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2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis
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recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to over 95% purity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83
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gene nfsA, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
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gene nfsA, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene nfsA, recombinant overexpression in Escherichia coli reporter strain SOS-R2, which contains a lacZ reporter gene under control of the SOS responsive promoter sfiA, functional nitroreductase NfsA expression in human HCT-116 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F42A
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site-directed mutagenesis, the mutant enzymes shows 99% reduced activity toward nitrofurazone compared to the wild-type, and loss of helix content
F42N
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site-directed mutagenesis, the mutant enzymes shows 96% reduced activity toward nitrofurazone compared to the wild-type, and loss of helix content
F42Y
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site-directed mutagenesis, the mutant enzymes shows 52% reduced activity toward nitrofurazone compared to the wild-type
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
environmental protection
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NfsA has potential applications in the biodegradation of nitroaromatic environment pollutants, e.g. explosives
medicine
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NfsA has potential applications in the anticancer strategy gene-directed enzyme prodrug therapy
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LINKS TO OTHER DATABASES (specific for EC-Number 1.7.1.B3)
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