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Literature summary for 1.7.1.B3 extracted from

  • Yang, J.; Zhan, J.; Bai, J.; Liu, P.; Xue, Y.; Yang, Q.
    Residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA (2013), Biotechnol. Lett., 35, 1693-1700 .
    View publication on PubMed
Search for more literature for 1.7.1.B3

Cloned(Commentary)

Cloned (Comment) Organism
gene nfsA, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha Escherichia coli

Protein Variants

Protein Variants Comment Organism
F42A site-directed mutagenesis, the mutant enzymes shows 99% reduced activity toward nitrofurazone compared to the wild-type, and loss of helix content Escherichia coli
F42N site-directed mutagenesis, the mutant enzymes shows 96% reduced activity toward nitrofurazone compared to the wild-type, and loss of helix content Escherichia coli
F42Y site-directed mutagenesis, the mutant enzymes shows 52% reduced activity toward nitrofurazone compared to the wild-type Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
additional information the enzyme is O2-insensitive Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten steady-state kinetics Escherichia coli
0.012
-
 nitrofurazone recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
0.013
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.02
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.022
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.023
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.029
-
 2,4,6-trinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
0.049
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.054
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.064
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.085
-
 NADPH recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
0.085
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.176
-
 2,4-dinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
0.206
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.313
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.322
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.327
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
0.332
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.366
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.487
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli P17117
-
-

Oxidation Stability

Oxidation Stability Organism
the enzyme is O2-insensitive Escherichia coli

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Escherichia coli

Reaction

Reaction Comment Organism Reaction ID
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 Escherichia coli
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.1
-
 purified recombinant mutant F46A, pH 7.0, 22°C, substrate nitrofurazone Escherichia coli
0.29
-
 purified recombinant mutant F46N, pH 7.0, 22°C, substrate nitrofurazone Escherichia coli
3.74
-
 purified recombinant mutant F46Y, pH 7.0, 22°C, substrate nitrofurazone Escherichia coli
7.75
-
 purified recombinant wild-type enzyme, pH 7.0, 22°C, substrate nitrofurazone Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,4,6-trinitrotoluene + NADPH + H+
-
 Escherichia coli ? + NADP+ + H2O
-
 ?
2,4-dinitrotoluene + NADPH + H+
-
 Escherichia coli ? + NADP+ + H2O
-
 ?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+ i.e. CB1954, an aziridinyl dinitrobenzamide prodrug Escherichia coli ? + NADP+
-
 ?
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 Escherichia coli ?
-
 ?
nitrofurazone + NADPH + H+
-
 Escherichia coli 5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
 ?

Subunits

Subunits Comment Organism
homodimer
-
 Escherichia coli

Synonyms

Synonyms Comment Organism
NfsA
-
 Escherichia coli
nitrofurazone reductase
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
22
-
 assay at room temperature Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.46
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.67
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.7
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.78
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
0.8
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
1.08
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
1.82
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
2.99
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
3.64
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
4.95
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
9.01
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
15.73
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
16.3
-
 NADPH recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
17.31
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
19.37
-
 nitrofurazone recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
24.27
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
24.94
-
 2,4-dinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
27.06
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
36.64
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
56.21
-
 2,4,6-trinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
FMN a flavoenzyme which contains FMN as the prosthetic group Escherichia coli
NADPH
-
 Escherichia coli

General Information

General Information Comment Organism
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 Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
2
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
5
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
9
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
10
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
12
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
16
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
50
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
75
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
80
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
84
-
 2,4-dinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
112
-
 5-(aziridin-1-yl)-2,4-dinitrobenzamide recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
142
-
 2,4-dinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
167
-
 NADPH recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
191.7
-
 NADPH recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
250
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
720
-
 nitrofurazone recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
1350
-
 2,4,6-trinitrotoluene recombinant mutant F46Y, pH 7.0, 22°C Escherichia coli
1610
-
 nitrofurazone recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli
1940
-
 2,4,6-trinitrotoluene recombinant wild-type enzyme, pH 7.0, 22°C Escherichia coli