Cloned (Comment) | Organism |
---|---|
gene nfsB, recombinant expression of wild-type and mutant N-terminally His6-tagged enzymes in Escherichia coli strain BL21(DE3) | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
F123A | site-directed mutagenesis, residue F123 is located in helix alpha6 constituting the substrate entry site, the mutant enzyme kinetics are altered compared to the wild-type enzyme | Escherichia coli |
F124A | site-directed mutagenesis, residue F124 is located in helix alpha6 constituting the substrate entry site, the mutant enzyme kinetics are altered compared to the wild-type enzyme | Escherichia coli |
F124N | site-directed mutagenesis, residue F124 is located in helix alpha6 constituting the substrate entry site, the mutant enzyme kinetics are altered compared to the wild-type enzyme | Escherichia coli |
F124W | site-directed mutagenesis, residue F124 is located in helix alpha6 constituting the substrate entry site, the mutant enzyme kinetics are altered compared to the wild-type enzyme | Escherichia coli |
N71S | site-directed mutagenesis, active site residue mutation, the mutant shows increased activity with prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) and nitrofurazone compared to the wild-type enzyme | Escherichia coli |
T41L | site-directed mutagenesis, active site residue mutation, the mutant shows increased activity with prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) and nitrofurazone compared to the wild-type enzyme | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten steady-state kinetics of recombinant His6-tagged wild-type and mutant enzymes | Escherichia coli | |
0.0023 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
0.0057 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
0.0072 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
0.0091 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
0.0174 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
0.0398 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
0.0534 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
0.0881 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
0.0928 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
0.0996 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
0.0997 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
0.163 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
0.172 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
0.265 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
0.306 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
0.339 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
0.38 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
0.411 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
0.518 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
0.575 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
0.732 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
1.069 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
1.168 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
1.495 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2,4,6-trinitrotoluene + NADH + H+ | Escherichia coli | - |
4-hydroxyamino-2,6-dinitrotoluene + NAD+ + H2O | - |
? | |
2,4,6-trinitrotoluene + NADPH + H+ | Escherichia coli | - |
4-hydroxyamino-2,6-dinitrotoluene + NADP+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P38489 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant N-terminally His6-tagged enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to over 95% purity | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2,4,6-trinitrotoluene + NADH + H+ | - |
Escherichia coli | 4-hydroxyamino-2,6-dinitrotoluene + NAD+ + H2O | - |
? | |
2,4,6-trinitrotoluene + NADPH + H+ | - |
Escherichia coli | 4-hydroxyamino-2,6-dinitrotoluene + NADP+ + H2O | - |
? | |
2,4,6-trinitrotoluene + NADPH + H+ | i.e. TNT, NfsB reduces TNT to the para isomer 4-hydroxyamino-2,6-dinitrotoluene more easily than the ortho isomer 2-hydroxyamino-4,6-dinitrotoluene | Escherichia coli | 4-hydroxyamino-2,6-dinitrotoluene + NADP+ + H2O | - |
? | |
2-amino-4,6-dinitrotoluene + NADH + H+ | - |
Escherichia coli | 4-hydroxyamino-2-aminotoluene + NAD+ + H2O | - |
? | |
4-amino-2,6-dinitrotoluene + NADH + H+ | - |
Escherichia coli | 2-hydroxyamino-4-aminotoluene + NAD+ + H2O | - |
? | |
additional information | no activity with 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene | Escherichia coli | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
NfsB | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.76 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
1.03 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
1.51 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
3.25 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
4.74 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
8.06 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
9.28 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
9.4 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
11.29 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
11.48 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
14.01 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
14.38 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
15.27 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
15.31 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
18.02 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
18.2 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
26.84 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
26.9 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
37.65 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
38.88 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
46.67 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
54.52 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
62.87 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
69.26 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | NfsB is capable of utilizing bothNADH and NADPH as cofactor | Escherichia coli | |
NADH | - |
Escherichia coli | |
NADPH | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | nitroreductases are a family of flavoproteins that catalyze the NAD(P)H-dependent reduction of nitro groups to hydroxyamino and/or amino groups on vari-ous nitro-substituted compounds | Escherichia coli |
additional information | the enzyme crystal structure of NfsB, PDB ID 1DS7, is used for molecular ligand docking studies | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
4 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
6 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
6 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
11 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
12 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
26 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
27 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
30 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
31 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
31 | - |
4-amino-2,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
45 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
46 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
57 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
65 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
181 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli | |
206 | - |
2-amino-4,6-dinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
290 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124N, with NADH | Escherichia coli | |
547 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124A, with NADH | Escherichia coli | |
653 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant N71S, with NADH | Escherichia coli | |
1177 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant T41L, with NADH | Escherichia coli | |
2170 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme, with NADH | Escherichia coli | |
2526 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F124W, with NADH | Escherichia coli | |
5111 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A, with NADH | Escherichia coli | |
5389 | - |
2,4,6-trinitrotoluene | pH 7.0, 37°C, recombinant His6-tagged mutant F123A/F124W, with NADH | Escherichia coli |