Information on EC 1.7.1.17 - FMN-dependent NADH-azoreductase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.7.1.17
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RECOMMENDED NAME
GeneOntology No.
FMN-dependent NADH-azoreductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
N,N-dimethyl-1,4-phenylenediamine, anthranilate:NAD+ oxidoreductase
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-(2-pyridylazo)-2-naphthol + 2 NADH + 2 H+
pyridin-2-amine + 1-aminonaphthalen-2-ol + 2 NAD+
show the reaction diagram
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-
-
?
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
show the reaction diagram
2-(4-dimethylaminophenylazo) benzoic acid + NADH + H+
N,N'-dimethyl-p-phenylenediamine + 2-aminobenzoic acid + NAD+
show the reaction diagram
4'-(diethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-diethyl-p-phenylenediamine + 2 NAD+
show the reaction diagram
i.e. ethyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
show the reaction diagram
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
show the reaction diagram
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
show the reaction diagram
7-nitrocoumarin-3-carboxylic acid + 2 NADH + 2 H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
show the reaction diagram
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via 7-nitrosocoumarin-3-carboxylic acid and 7-hydroxylaminocoumarin-3-carboxylic acid intermediates
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-
?
7-nitrocoumarin-3-carboxylic acid + NADH + H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
show the reaction diagram
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-
-
-
?
acid red 88 + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NAD+
show the reaction diagram
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NAD+
show the reaction diagram
amaranth + 2 NADPH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NADP+
show the reaction diagram
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-
-
-
?
congo red + 2 NADH + 2 H+
? + 2 NAD+
show the reaction diagram
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-
-
?
ethyl red + 2 NADH + 2 H+
anthranilate + N,N-diethyl-1,4-phenylenediamine + 2 NAD+
show the reaction diagram
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-
-
-
?
fast red E + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 5-amino-6-hydroxynaphthalene-2-sulfonic acid + 2 NAD+
show the reaction diagram
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-
-
-
?
fast red E + 2 NADPH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 5-amino-6-hydroxynaphthalene-2-sulfonic acid + 2 NADP+
show the reaction diagram
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-
-
-
?
menadione + 2 NADH + 2 H+
? + 2 NAD+
show the reaction diagram
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-
-
-
?
orange G + 2 NADH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NAD+
show the reaction diagram
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-
-
-
?
orange G + 2 NADPH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NADP+
show the reaction diagram
orange I + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 4-aminonaphthalen-1-ol + 2 NAD+
show the reaction diagram
orange II + 2 NADPH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NADP+ + 2 NAD+
show the reaction diagram
ponceau BS + 2 NADH + 2 H+
? + 2 NAD+
show the reaction diagram
ponceau S + 2 NADH + 2 H+
? + 2 NAD+
show the reaction diagram
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-
-
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
show the reaction diagram
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
show the reaction diagram
7-nitrocoumarin-3-carboxylic acid + NADH + H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
show the reaction diagram
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-
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
roseoflavin mononucleotide
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roseoflavin mononucleotide binds to the AzoR apoenzyme with an even higher affinity compared to that of the natural cofactor FMN. Roseoflavin mononucleotide binding does not affect the overall topology of the enzyme and also does not interfere with dimerization of AzoR. With roseoflavin mononucleotide, the holoenzyme shiws about 30% of wild-type activity
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
A0A1B1KJ01
43% activation at 0.05 mM
Mn2+
A0A1B1KJ01
119% activation at 0.05 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
A0A1B1KJ01
10% inhibition at 0.05 mM
dicoumarol
Fe2+
A0A1B1KJ01
13% inhibition at 0.05 mM
Fe3+
A0A1B1KJ01
14% inhibition at 0.05 mM
Mg2+
A0A1B1KJ01
8% inhibition at 0.05 mM
Zn2+
A0A1B1KJ01
16% inhibition at 0.05 mM
additional information
A0A1B1KJ01
the enzyme is oxygen-insensitive
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0179
2-(4-dimethylaminophenyl)diazenylbenzoate
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recombinant enzyme, pH 7.4, 30C
0.002 - 0.034
4'-(dimethylamino)-azobenzene-2-carboxylic acid
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0.034
Amaranth
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pH 7.4, 37C
0.0107 - 0.371
NADH
15
NADPH
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pH 7.2, 25C
additional information
additional information
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.626
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cofactor NADPH, pH 7.0, 25C
7.3
substrate acid red 88, pH 7.4, 30C
11.4
substrate orange I, pH 7.4, 30C
18
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substrate orange G, cofactor NADH, pH 7.4, 37C
22
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substrate amaranth, cofactor NADPH, pH 7.4, 37C
24.1
substrate 1-(2-pyridylazo)-2-naphthol,pH 7.4, 30C
25
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substrate amaranth, cofactor FADH2, pH 7.4, 37C
25.8
30C, pH 7.4, substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid
26
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substrate amaranth, cofactor FMNH2, pH 7.4, 37C
26.6
substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid, pH 7.4, 30C
63
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substrate fast red E, cofactor NADH, pH 7.4, 37C
63.9
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pH 7.4, 30C
74
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substrate amaranth, cofactor NADH, pH 7.4, 37C
98.9
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purified recombinant enzyme, pH 7.4, 30C, methyl red with FAD
137
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substrate amaranth, pH 7.1, 23C
139
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substrate orange II, pH 7.1, 23C
141.25
A0A1B1KJ01
purified recombinant enzyme, pH 4.0, 25C
167
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substrate ponceau S, pH 7.1, 23C
181
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substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid, pH 7.1, 23C
188.6
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purified recombinant enzyme, pH 7.4, 30C, methyl red with FMN
362.6
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purified recombinant enzyme, pH 7.4, 30C, menadione with FMN
441
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purified recombinant enzyme, pH 7.4, 30C, ethyl red with FMN
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
A0A1B1KJ01
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7
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
35
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assay at
53
A0A1B1KJ01
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 50
A0A1B1KJ01
the enzyme activity drops largely after 50C, and there is no activity left when temperature rises to 70C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.92
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sequence calculation
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
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gel filtration and PAGE
42000
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gel filtration; gel fitlration
43000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 29000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
docking simulations. The isoalloxazine ring of FAD localizes at the same site and plays the same role as that of FMN in AzrA
in complex with FMN, to 2.07 A resolution. The AzoA monomer shows a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind
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purified oxidized or reduced AzoR, free or in complex with inhibitor dicoumarol, hanging drop vapor diffusion method, mixing of equal volumes of 8 mg/ml protein in 10 mM Tris-HCl, pH 8.0, 100 mM NAD+ and 0.1 mM FMN with reservoir solution containing 200 mM NaOAc, 200 mM sodium cacodylate, pH 6.7, 15% w/v PEG 8000, and 3% v/v dimethyl sulfoxide, equilibration over reservoir solution, at 25 C, 2 weeks, X-ray diffraction structure determination and analysis at 1.4-2.3 A resolution, molecular replacement using the 1.8 resolution structure of oxidized AzoR as a search model, modelling
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purified recombinant enzyme, sitting drop vapour-diffusion method, mixing 0.015 ml of 23 mg/ml protein in 10 mM Tris-HCl, pH 8.0, and 1 mM FMN, with an equal volume of reservoir solution containing 200 mM MgCl2, 30% v/v 2-propanol, and 100 mM HEPES, pH 7.5, equilibration over 0.5 ml reservoir solution, one week, 15C, method optimization, crystal soaking in heavy metal solution with K2PtCl4, X-ray diffraction structure determination and analysis at 1.8-2.5 A resolution. FMN is tightly bound to the protein moiety, and this interaction is essential for the crystallization of AzoR
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to 1.6 A resolution, space group F222, with unit-cell parameters a = 72.1, b = 95.5, c = 146.1 A
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
30C, 1 h, more than 90% of maximum activity
744136
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 40
A0A1B1KJ01
purified recombinant enzyme, pH 7.0, 0.1 M sodium phosphate buffer, 30 min, stable at
45
stable for at least 1 h
55
A0A1B1KJ01
purified recombinant enzyme, unfolding temperature
70
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stable for 30 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity is oxygen insensitive
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is oxygen-insensitive
A0A1B1KJ01
743106
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; native enzyme 4500fold by anion exchange chromatography, Blue Sepharose affinity chromtography, gel filtration, hydroxyapatite chromatography, again anion exchange chromatography and hydrophobic interaction chromatography. Recombinant enzyme AcpD from Escherichia coli, strain JM109 by anion exchange chromatography, Blue Sepharose affinity chromtography, gel filtration, and chromatography on GIGAPITE resin, the purified gene product exhibits activity corresponding to that of the native azoreductase
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
A0A1B1KJ01
recombinant His-tagged enzyme from Escherichia coli strain XL1-Blue by nickel affinity chromatography
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recombinant His-tagged wild-type and mutant AzoR enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli; gene acpD, sequence comparisons, recombinant overexpression of the gene provides Escherichia coli strain JM109 with more than 800 times increased azoreductase activity
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gene azoR, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic tree, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
A0A1B1KJ01
gene azoR, recombinant expression of His-tagged wild-type and mutant AzoR enzymes in Escherichia coli BL21(DE3)
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gene azoR, recombinant overexpression of His-tagged enzyme in Escherichia coli strain XL1-Blue
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N121Q
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115% of wild-type activity
R21G/N121A
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136% of wild-type activity
R21G/N121Q
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171% of wild-type activity
R21K
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80% of wild-type activity
R21K/N121A
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41% of wild-type activity
R21K/N121Q
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40% of wild-type activity
W105A
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complete loss of both affinity for FMN and enzyme activity
W105F
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31fold decrease in vmax value, Km value similar to wild-type
W105G
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complete loss of both affinity for FMN and enzyme activity
W105H
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8fold decrease in vmax value, Km value similar to wild-type
W105Q
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68fold decrease in vmax value, Km value similar to wild-type
W105Y
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22% reduction in vmax value
N121Q
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115% of wild-type activity
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R21G/N121A
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136% of wild-type activity
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R21G/N121Q
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171% of wild-type activity
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R21K
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80% of wild-type activity
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R21K/N121A
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41% of wild-type activity
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W105A
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complete loss of both affinity for FMN and enzyme activity
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W105F
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31fold decrease in vmax value, Km value similar to wild-type
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W105H
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8fold decrease in vmax value, Km value similar to wild-type
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W105Q
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68fold decrease in vmax value, Km value similar to wild-type
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W105Y
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22% reduction in vmax value
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F162A
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site-directed mutagenesis
R59A
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site-directed mutagenesis, the mutation enhances the Vmax value for p-methyl red 27fold with a 3.8fold increase of the Km value, residue Arg59 decides the substrate specificity of AzoR
R59G
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mutation influences the formation of dilution-induced intermediates. Mutant R59G contains only two types of FMN, emitting at 530 nm and 600 nm
Y120A
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site-directed mutagenesis
additional information
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deletion of the azoR gene in Escherichia coli strain MG1655 has no strong influence on the 7NCCA reduction of the cells even though the pure enzyme displays a clear nitroreductase activity with this compound