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Literature summary for 1.7.1.B2 extracted from

  • Bai, J.; Yang, J.; Liu, P.; Yang, Q.
    Transformation pathway of 2,4,6-trinitrotoluene by Escherichia coli nitroreductases and improvement of activity using structure-based mutagenesis (2015), Process Biochem., 50, 705-711 .
    View publication on PubMed
Search for more literature for 1.7.1.B2

Cloned(Commentary)

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

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 [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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Escherichia coli P38489
-
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
NfsB
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Escherichia coli

Turnover Number [1/s]

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

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

Cofactor

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

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 [mM/s]

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