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 | 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 | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme is O2-insensitive | Escherichia coli |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P17117 | - |
- |
Oxidation Stability | Organism |
---|---|
the enzyme is O2-insensitive | Escherichia coli |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Escherichia coli |
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 |
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 | 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 | Comment | Organism |
---|---|---|
homodimer | - |
Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
NfsA | - |
Escherichia coli |
nitrofurazone reductase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Escherichia coli |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FMN | a flavoenzyme which contains FMN as the prosthetic group | Escherichia coli | |
NADPH | - |
Escherichia coli |
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 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 |