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an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+
an aromatic amine + NADP+ + H2O = an aromatic hydroxylamine + NADPH + H+
(1a)
-
-
-
an aromatic hydroxylamine + NADP+ = an aromatic nitric oxide + NADPH + H+
(1b)
-
-
-
an aromatic nitric oxide + NADP+ + H2O = an aromatic nitrate + NADPH + H+
(1c)
-
-
-
an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+

(overall reaction)
-
-
-
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
-
-
-
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
-
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
-
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
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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2,4,6-trinitrophenyl N-methylnitramine + 4 NADPH + H+
?
-
-
-
-
?
2,4,6-trinitrotoluene + 2 NADPH + H+
?
-
-
-
-
?
2,4,6-trinitrotoluene + NADPH + H+
4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
2,4,6-trinitrotoluene + NADPH + H+
? + NADP+ + H2O
2,4-dinitrotoluene + NADPH + H+
? + NADP+ + H2O
-
-
-
?
2-amino-4,6-dinitrotoluene + NADPH + H+
? + NADP+ + H2O
-
-
-
?
4-amino-2,6-dinitrotoluene + NADPH + H+
? + NADP+ + H2O
-
-
-
?
4-nitroacetophenone + NADPH + H+
?
-
-
-
-
?
4-nitrobenzoic acid + NADPH + H+
?
-
-
-
-
?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+
? + NADP+
7-nitrocoumarin + 3 NADPH + 3 H+
7-aminocoumarin + 3 NADP+
m-dinitrobenzene + NADPH + H+
? + NADP+
-
-
-
-
?
methyl 4-nitrobenzoate + NADPH + H+
? + NADP+ + H2O
-
-
-
-
?
nifuroxime + NADPH + H+
? + NADP+
-
-
-
-
?
nitro-CBI-5-[(dimethylamino)ethoxy]indole + NAD(P)H + H+
? + NAD(P)+
a nitro-CBI prodrug, low activity
-
-
?
nitrobenzene + NADPH + H+
?
-
-
-
-
?
nitrobenzene + NADPH + H+
? + NADP+
-
-
-
-
?
nitrofurantoin + NADPH + H+
? + NADP+
-
-
-
-
?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
o-dinitrobenzene + NADPH + H+
? + NADP+
-
-
-
-
?
p-dinitrobenzene + NADPH + H+
? + NADP+
-
-
-
-
?
p-nitroacetophenone + NADPH + H+
? + NADP+
-
-
-
-
?
p-nitrobenzaldehyde + NADPH + H+
? + NADP+
-
-
-
-
?
p-nitrobenzoic acid + NADPH + H+
? + NADP+
-
-
-
-
?
tetryl + NADPH + H+
? + NADP+
-
-
-
-
?
additional information
?
-
2,4,6-trinitrotoluene + NADPH + H+

4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
-
-
-
?
2,4,6-trinitrotoluene + NADPH + H+
4-hydroxylamino-2,6-dinitrotoluene + NADP+ + H2O
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
-
-
?
2,4,6-trinitrotoluene + NADPH + H+

? + NADP+ + H2O
-
-
-
-
?
2,4,6-trinitrotoluene + NADPH + H+
? + NADP+ + H2O
-
-
-
?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+

? + NADP+
-
i.e. CB1954, an aziridinyl dinitrobenzamide prodrug
-
-
?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+
? + NADP+
i.e. CB1954, an aziridinyl dinitrobenzamide prodrug
-
-
?
7-nitrocoumarin + 3 NADPH + 3 H+

7-aminocoumarin + 3 NADP+
-
-
-
?
7-nitrocoumarin + 3 NADPH + 3 H+
7-aminocoumarin + 3 NADP+
-
-
-
-
?
nitrofurazone + NADPH + H+

5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
-
?
nitrofurazone + NADPH + H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
?
additional information

?
-
-
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
-
-
?
additional information
?
-
an intrinsically oxygen-insensitive nature of the two-electron reduction mechanism
-
-
?
additional information
?
-
-
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
-
-
?
additional information
?
-
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
-
-
?
additional information
?
-
no activity with 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene
-
-
?
additional information
?
-
coenzyme Q and tetracycline may be used as electron acceptors, but with much lower specific activities than menadione, cf EC 1.6.5.5. Nitro compounds dinitrotoluene, 7-nitrocoumarin, and metronidazole can be used as electron acceptors
-
-
-
additional information
?
-
-
coenzyme Q and tetracycline may be used as electron acceptors, but with much lower specific activities than menadione, cf EC 1.6.5.5. Nitro compounds dinitrotoluene, 7-nitrocoumarin, and metronidazole can be used as electron acceptors
-
-
-
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0.013 - 0.033
2,4,6-trinitrotoluene
0.176 - 0.366
2,4-dinitrotoluene
0.3257
2-amino-4,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
0.6427
4-amino-2,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
0.23 - 0.487
5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.046
m-dinitrobenzene
-
pH 7.0, 25°C
0.031
nifuroxime
-
pH 7.0, 25°C
1.5
nitrobenzene
-
pH 7.0, 25°C
0.019
Nitrofurantoin
-
pH 7.0, 25°C
0.012 - 0.064
nitrofurazone
0.018
o-dinitrobenzene
-
pH 7.0, 25°C
0.037
p-dinitrobenzene
-
pH 7.0, 25°C
0.39
p-nitroacetophenone
-
pH 7.0, 25°C
0.17
p-nitrobenzaldehyde
-
pH 7.0, 25°C
2.5
p-nitrobenzoic acid
-
pH 7.0, 25°C
0.011
tetryl
-
pH 7.0, 25°C
additional information
additional information
-
0.013
2,4,6-trinitrotoluene

recombinant mutant F46Y, pH 7.0, 22°C
0.02
2,4,6-trinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
0.029
2,4,6-trinitrotoluene
recombinant wild-type enzyme, pH 7.0, 22°C
0.029
2,4,6-trinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
0.033
2,4,6-trinitrotoluene
-
pH 7.0, 25°C
0.176
2,4-dinitrotoluene

recombinant wild-type enzyme, pH 7.0, 22°C
0.206
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
0.313
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
0.366
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
0.23
5-(aziridin-1-yl)-2,4-dinitrobenzamide

-
pH 7.0, 25°C
0.322
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
0.327
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant wild-type enzyme, pH 7.0, 22°C
0.332
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
0.487
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
0.00085
NADPH

-
pH 7.0, 23°C, recombinant enzyme, with nitrofurazone
0.049
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
0.054
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
0.085
NADPH
recombinant wild-type enzyme, pH 7.0, 22°C
0.085
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
0.012
nitrofurazone

recombinant wild-type enzyme, pH 7.0, 22°C
0.0163
nitrofurazone
-
pH 7.0, 23°C, recombinant enzyme
0.022
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
0.023
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
0.064
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
additional information
additional information

Michaelis-Menten steady-state kinetics
-
additional information
additional information
-
enzyme kinetic analysis, overview
-
additional information
additional information
Michaelis-Menten steady-state kinetics of recombinant His6-tagged enzyme
-
additional information
additional information
-
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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110
2,4,6-trinitrophenyl N-methylnitramine
-
at pH 7.0 and 25°C
-
0.67 - 135.1
2,4,6-trinitrotoluene
0.7 - 24.94
2,4-dinitrotoluene
12.36
2-amino-4,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
1.05
4-amino-2,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
59.4
4-Nitroacetophenone
-
at pH 7.0 and 25°C
64.3
4-nitrobenzoic acid
-
at pH 7.0 and 25°C
1.08 - 36.64
5-(aziridin-1-yl)-2,4-dinitrobenzamide
55
m-dinitrobenzene
-
pH 7.0, 25°C
180
nifuroxime
-
pH 7.0, 25°C
136
Nitrofurantoin
-
pH 7.0, 25°C
0.8 - 19.37
nitrofurazone
60
o-dinitrobenzene
-
pH 7.0, 25°C
27
p-dinitrobenzene
-
pH 7.0, 25°C
59
p-nitroacetophenone
-
pH 7.0, 25°C
30
p-nitrobenzaldehyde
-
pH 7.0, 25°C
64
p-nitrobenzoic acid
-
pH 7.0, 25°C
0.67
2,4,6-trinitrotoluene

recombinant mutant F46Y, pH 7.0, 22°C
4.95
2,4,6-trinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
27.06
2,4,6-trinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
56.2
2,4,6-trinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
56.21
2,4,6-trinitrotoluene
recombinant wild-type enzyme, pH 7.0, 22°C
89
2,4,6-trinitrotoluene
-
pH 7.0, 25°C
135.1
2,4,6-trinitrotoluene
-
at pH 7.0 and 25°C
0.7
2,4-dinitrotoluene

recombinant mutant F46Y, pH 7.0, 22°C
3.64
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
17.31
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
24.94
2,4-dinitrotoluene
recombinant wild-type enzyme, pH 7.0, 22°C
1.08
5-(aziridin-1-yl)-2,4-dinitrobenzamide

recombinant mutant F46Y, pH 7.0, 22°C
2.99
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
15
5-(aziridin-1-yl)-2,4-dinitrobenzamide
-
pH 7.0, 25°C
24.27
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
36.64
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant wild-type enzyme, pH 7.0, 22°C
0.46
NADPH

recombinant mutant F46Y, pH 7.0, 22°C
0.78
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
9.01
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
16.3
NADPH
recombinant wild-type enzyme, pH 7.0, 22°C
14
nitrobenzene

-
pH 7.0, 25°C
14.2
nitrobenzene
-
at pH 7.0 and 25°C
0.8
nitrofurazone

recombinant mutant F46Y, pH 7.0, 22°C
1.82
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
15.73
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
19.37
nitrofurazone
recombinant wild-type enzyme, pH 7.0, 22°C
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7900
2,4,6-trinitrophenyl N-methylnitramine
-
at pH 7.0 and 25°C
-
50 - 2700
2,4,6-trinitrotoluene
2 - 142
2,4-dinitrotoluene
38
2-amino-4,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
1.6
4-amino-2,6-dinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
150
4-Nitroacetophenone
-
at pH 7.0 and 25°C
2.5
4-nitrobenzoic acid
-
at pH 7.0 and 25°C
2 - 112
5-(aziridin-1-yl)-2,4-dinitrobenzamide
1195.7
m-dinitrobenzene
-
pH 7.0, 25°C
5806.5
nifuroxime
-
pH 7.0, 25°C
7157.9
Nitrofurantoin
-
pH 7.0, 25°C
3333.3
o-dinitrobenzene
-
pH 7.0, 25°C
729.7
p-dinitrobenzene
-
pH 7.0, 25°C
151.3
p-nitroacetophenone
-
pH 7.0, 25°C
176.5
p-nitrobenzaldehyde
-
pH 7.0, 25°C
25.6
p-nitrobenzoic acid
-
pH 7.0, 25°C
7727.27
tetryl
-
pH 7.0, 25°C
50
2,4,6-trinitrotoluene

recombinant mutant F46Y, pH 7.0, 22°C
250
2,4,6-trinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
1350
2,4,6-trinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
1931
2,4,6-trinitrotoluene
pH 7.0, 37°C, recombinant His6-tagged enzyme
1940
2,4,6-trinitrotoluene
recombinant wild-type enzyme, pH 7.0, 22°C
2697
2,4,6-trinitrotoluene
-
pH 7.0, 25°C
2700
2,4,6-trinitrotoluene
-
at pH 7.0 and 25°C
2
2,4-dinitrotoluene

recombinant mutant F46Y, pH 7.0, 22°C
12
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
84
2,4-dinitrotoluene
recombinant mutant F46Y, pH 7.0, 22°C
142
2,4-dinitrotoluene
recombinant wild-type enzyme, pH 7.0, 22°C
2
5-(aziridin-1-yl)-2,4-dinitrobenzamide

recombinant mutant F46Y, pH 7.0, 22°C
9
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
65.2
5-(aziridin-1-yl)-2,4-dinitrobenzamide
-
pH 7.0, 25°C
75
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant mutant F46Y, pH 7.0, 22°C
112
5-(aziridin-1-yl)-2,4-dinitrobenzamide
recombinant wild-type enzyme, pH 7.0, 22°C
5
NADPH

recombinant mutant F46Y, pH 7.0, 22°C
16
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
167
NADPH
recombinant mutant F46Y, pH 7.0, 22°C
191.7
NADPH
recombinant wild-type enzyme, pH 7.0, 22°C
1.5
nitrobenzene

-
at pH 7.0 and 25°C
9.3
nitrobenzene
-
pH 7.0, 25°C
10
nitrofurazone

recombinant mutant F46Y, pH 7.0, 22°C
80
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
720
nitrofurazone
recombinant mutant F46Y, pH 7.0, 22°C
1610
nitrofurazone
recombinant wild-type enzyme, pH 7.0, 22°C
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evolution

-
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
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
-
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
-
physiological function

-
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
residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA, Phe42 is important for maintaining NfsA activity and structure
physiological function
enzyme is a flavoprotein with NADPH-dependent quinone reductase and nitroreductase activities
physiological function
-
enzyme is a flavoprotein with NADPH-dependent quinone reductase and nitroreductase activities
-
additional information

-
comparisons of the quantitative structure-activity relationships of single-electron reduction of quinones and nitroaromatic compounds by dehydrogenase (electron transferase) flavoenzymes, overview
additional information
the enzyme crystal structure of NfsA, PDB ID 1F5V, is used for molecular ligand docking studies
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