BRENDA - Enzyme Database show
show all sequences of 1.7.1.B3

Purification and characetrizatio of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase

Zenno, S.; Kobori, T.; Tanokura, M.; Saigo, K.; Biosci. Biotechnol. Biochem. 62, 1978-1987 (1998)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83
Bacillus subtilis
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
enzyme kinetic analysis, overview
Bacillus subtilis
0.00085
-
NADPH
pH 7.0, 23°C, recombinant enzyme, with nitrofurazone
Bacillus subtilis
0.0163
-
nitrofurazone
pH 7.0, 23°C, recombinant enzyme
Bacillus subtilis
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
53000
-
recombinant untagged enzyme, gel filtration
Bacillus subtilis
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
nitrofurazone + NADPH + H+
Bacillus subtilis
-
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Bacillus subtilis
-
-
-
Bacillus subtilis ISW 1214
-
-
-
Purification (Commentary)
Commentary
Organism
recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis
Bacillus subtilis
Reaction
Reaction
Commentary
Organism
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
Bacillus subtilis
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
52.5
-
purified recombinant untagged enzyme, pH 7.0, 23°C
Bacillus subtilis
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
methyl 4-nitrobenzoate + NADPH + H+
-
742109
Bacillus subtilis
? + 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
742109
Bacillus subtilis
?
-
-
-
-
nitrofurazone + NADPH + H+
-
742109
Bacillus subtilis
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
?
Subunits
Subunits
Commentary
Organism
homodimer
2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
Bacillus subtilis
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
23
-
assay at
Bacillus subtilis
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Bacillus subtilis
Cofactor
Cofactor
Commentary
Organism
Structure
FMN
tightly associated to, 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
Bacillus subtilis
additional information
NADPH is much more effective than NADH
Bacillus subtilis
NADPH
dependent on
Bacillus subtilis
Cloned(Commentary) (protein specific)
Commentary
Organism
gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83
Bacillus subtilis
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
FMN
tightly associated to, 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
Bacillus subtilis
additional information
NADPH is much more effective than NADH
Bacillus subtilis
NADPH
dependent on
Bacillus subtilis
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
enzyme kinetic analysis, overview
Bacillus subtilis
0.00085
-
NADPH
pH 7.0, 23°C, recombinant enzyme, with nitrofurazone
Bacillus subtilis
0.0163
-
nitrofurazone
pH 7.0, 23°C, recombinant enzyme
Bacillus subtilis
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
53000
-
recombinant untagged enzyme, gel filtration
Bacillus subtilis
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
nitrofurazone + NADPH + H+
Bacillus subtilis
-
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis
Bacillus subtilis
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
52.5
-
purified recombinant untagged enzyme, pH 7.0, 23°C
Bacillus subtilis
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
methyl 4-nitrobenzoate + NADPH + H+
-
742109
Bacillus subtilis
? + 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
742109
Bacillus subtilis
?
-
-
-
-
nitrofurazone + NADPH + H+
-
742109
Bacillus subtilis
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
homodimer
2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
Bacillus subtilis
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
23
-
assay at
Bacillus subtilis
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Bacillus subtilis
General Information
General Information
Commentary
Organism
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
Bacillus subtilis
General Information (protein specific)
General Information
Commentary
Organism
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
Bacillus subtilis
Other publictions for EC 1.7.1.B3
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
741786
Valiauga
Reduction of quinones and nit ...
Escherichia coli
Arch. Biochem. Biophys.
614
14-22
2017
-
2
-
-
-
-
2
13
-
-
-
-
-
1
-
-
-
1
-
-
-
-
13
-
1
-
-
12
1
-
-
2
2
-
-
-
2
-
2
-
-
-
-
2
2
13
-
-
-
-
-
-
-
-
-
-
-
-
13
-
1
-
-
12
1
-
-
-
-
2
2
-
12
12
743709
Bai
Transformation pathway of 2,4 ...
Escherichia coli
Process Biochem.
50
705-711
2015
-
-
1
-
-
-
-
4
-
-
-
1
-
1
-
-
1
-
-
-
-
-
5
-
1
-
-
3
1
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
4
-
-
-
1
-
-
-
1
-
-
-
-
5
-
1
-
-
3
1
-
-
-
-
2
2
-
3
3
742167
Yang
Residue Phe42 is critical for ...
Escherichia coli
Biotechnol. Lett.
35
1693-1700
2013
-
-
1
-
3
-
1
19
-
-
-
-
-
1
1
-
1
1
-
-
4
-
5
1
1
-
-
20
1
-
-
2
-
-
-
-
-
1
2
-
3
-
-
1
-
19
-
-
-
-
-
1
-
1
-
-
4
-
5
1
1
-
-
20
1
-
-
-
-
1
1
-
20
20
743280
Green
Pseudomonas aeruginosa NfsB a ...
Escherichia coli
Mol. Cancer
12
58
2013
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
742109
Zenno
Purification and characetriza ...
Bacillus subtilis, Bacillus subtilis ISW 1214
Biosci. Biotechnol. Biochem.
62
1978-1987
1998
-
-
1
-
-
-
-
3
-
-
1
1
-
2
-
-
1
1
-
-
1
-
3
1
1
-
-
-
1
-
-
3
-
-
-
-
-
1
3
-
-
-
-
-
-
3
-
-
1
1
-
-
-
1
-
-
1
-
3
1
1
-
-
-
1
-
-
-
-
1
1
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-
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