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Information on EC 1.7.1.17 - FMN-dependent NADH-azoreductase
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1.7.1.17 FMN-dependent NADH-azoreductaseIUBMB Comments
Requires FMN. The enzyme catalyses the reductive cleavage of an azo bond in aromatic azo compounds to form the corresponding amines. Does not accept NADPH. cf. EC 1.7.1.6, azobenzene reductase.
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The enzyme appears in viruses and cellular organisms
Reaction Schemes
Synonyms
acpD, azo-dye reductase, AzoA, AzoR, AzoR1, azoreductase, AzoRo, AzrA, AzrG, flavin-containing oxygen-insensitive azoreductase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
azo-dye reductase
AzoA
AzoR
AzoR1
AzoRo
AzrA
flavin-containing oxygen-insensitive azoreductase
FMN-dependent NADH-azoreductase
NADH-azoreductase
-
-
ambiguous
-
AzoR
-
-
ambiguous
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
-
-
-
-
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
ping-pong reaction mechanism
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
ping-pong reaction mechanism, overview
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
ping-pong reaction mechanism. Enzymatic reaction involves an electron transfer from nicotinamide dinucleotide (NADH) to flavin mononucleotide (FMN) (ping) followed by a transfer from FMNH2 to the substrate (pong), key role of flavin
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SYSTEMATIC NAME
IUBMB Comments
N,N-dimethyl-1,4-phenylenediamine, anthranilate:NAD+ oxidoreductase
Requires FMN. The enzyme catalyses the reductive cleavage of an azo bond in aromatic azo compounds to form the corresponding amines. Does not accept NADPH. cf. EC 1.7.1.6, azobenzene reductase.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
1-(2-pyridylazo)-2-naphthol + 2 NADH + 2 H+
pyridin-2-amine + 1-aminonaphthalen-2-ol + 2 NAD+
-
-
-
?
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
2-(4-dimethylaminophenylazo) benzoic acid + NADH + H+
N,N'-dimethyl-p-phenylenediamine + 2-aminobenzoic acid + NAD+
4'-(diethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-diethyl-p-phenylenediamine + 2 NAD+
i.e. ethyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
7-nitrocoumarin-3-carboxylic acid + 2 NADH + 2 H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
via 7-nitrosocoumarin-3-carboxylic acid and 7-hydroxylaminocoumarin-3-carboxylic acid intermediates
-
-
?
7-nitrocoumarin-3-carboxylic acid + NADH + H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
-
-
-
?
acid red 88 + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NAD+
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NAD+
amaranth + 2 NADPH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NADP+
-
-
-
-
?
ethyl red + 2 NADH + 2 H+
anthranilate + N,N-diethyl-1,4-phenylenediamine + 2 NAD+
-
-
-
?
fast red E + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 5-amino-6-hydroxynaphthalene-2-sulfonic acid + 2 NAD+
-
-
-
-
?
fast red E + 2 NADPH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 5-amino-6-hydroxynaphthalene-2-sulfonic acid + 2 NADP+
-
-
-
-
?
orange G + 2 NADH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NAD+
-
-
-
-
?
orange G + 2 NADPH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NADP+
orange II + 2 NADPH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NADP+ + 2 NAD+
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
i.e. azo dye methyl red
-
-
?
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
i.e. azo dye methyl red, via a diazene intermediate
-
-
?
2-(4-dimethylaminophenylazo) benzoic acid + NADH + H+
N,N'-dimethyl-p-phenylenediamine + 2-aminobenzoic acid + NAD+
i.e. methyl red
-
-
?
2-(4-dimethylaminophenylazo) benzoic acid + NADH + H+
N,N'-dimethyl-p-phenylenediamine + 2-aminobenzoic acid + NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
-
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
-
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
-
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
i.e. methyl red
-
-
?
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADPH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NADP+
-
i.e. methyl red
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
-
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
i.e. methyl yellow
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
-
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
i.e. methyl yellow
-
-
?
acid red 88 + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NAD+
-
-
-
?
acid red 88 + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NAD+
-
-
-
?
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NAD+
-
-
-
?
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NAD+
-
-
-
?
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 2 NAD+
-
-
-
-
?
orange G + 2 NADPH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NADP+
-
-
-
?
orange G + 2 NADPH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NADP+
-
-
-
?
orange G + 2 NADPH + 2 H+
aniline + 8-amino-7-hydroxynaphthalene-1,3-disulfonic acid + 2 NADP+
-
-
-
-
?
orange I + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 4-aminonaphthalen-1-ol + 2 NAD+
-
-
-
?
orange I + 2 NADH + 2 H+
4-aminobenzene-1-sulfonic acid + 4-aminonaphthalen-1-ol + 2 NAD+
-
-
-
?
orange II + 2 NADPH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NADP+ + 2 NAD+
-
-
-
?
orange II + 2 NADPH + 2 H+
4-aminobenzene-1-sulfonic acid + 1-aminonaphthalen-2-ol + 2 NADP+ + 2 NAD+
-
-
-
?
additional information
?
-
active site structure analysis and substrate specificity, Azo compound binding structure, overview
-
-
?
additional information
?
-
AzoR utilizes NADH but not NADPH as an electron donor and binds FMN as a flavin cofactor
-
-
?
additional information
?
-
no activity with Ponceau SX. Recombinant AcpD reaction follows a ping-pong mechanism requiring 2 mol of NADH to reduce 1 mol of methyl red into 2-aminobenzoic acid and N,N'-dimethyl-p-phenylenediamine. On the other hand, the gene product cannot convert holo-acyl carrier protein into the apo form under either in vitro or in vivo conditions. These data indicate that the acpD gene product is not acyl carrier protein phosphodiesterase but an azoreductase
-
-
?
additional information
?
-
-
enzyme additionally acts as nitroreductase and selectively reduces the nitro group of 2-nitrophenol, 4-nitrobenzoic acid, 2-nitro-benzaldehyde and 3-nitrophenol
-
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
i.e. azo dye methyl red
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
7-nitrocoumarin-3-carboxylic acid + NADH + H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
-
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
-
-
-
?
4-(dimethylamino)azobenzene + NADH + H+
N,N-dimethyl-1,4-phenylenediamine + aniline + NAD+
-
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
roseoflavin mononucleotide
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
FAD
60% of the activity with FMN. FAD is stably retained in the enzyme molecule without degradation to FMN
FMN
a flavin-containing enzyme the enzyme binds 1 FMN per subunit
FMN
FMN is tightly bound to the protein moiety, and this interaction is essential for the crystallization of AzoR
FMN
-
coexistence of at least three FMN-containing components in wild-type AzoR, i.e. FMN emitting at 490 nm, 530 nm and 600 nm, respectively
FMN
residues Trp105, Asn106, Leu107, Gly150, Gly151, Tyr153, Asn121 and Tyr129 are involved in binding the FMN isoalloxazine ring. Residue Trp105 is involved in FMN stabilization and destabilization
NADH
reaction with NADH is 13fold faster than with NADPH
NADPH
reaction with NADH is 13fold faster than with NADPH
additional information
AzoR utilizes NADH but not NADPH as an electron donor and binds FMN as a flavin cofactor
-
additional information
no cofactor: 8-amino-8-demethyl-riboflavin mononucleotide
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0179
2-(4-dimethylaminophenyl)diazenylbenzoate
recombinant enzyme, pH 7.4, 30°C
0.002
4'-(dimethylamino)-azobenzene-2-carboxylic acid
cofactor roseoflavin mononucleotide, pH 7.4, 30°C
0.011
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant R21G/N121A, pH 7.1, temperature not specified in the publication
0.016
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant R21G/N121Q, pH 7.1, temperature not specified in the publication
0.02
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant R21K, pH 7.1, temperature not specified in the publication
0.024
4'-(dimethylamino)-azobenzene-2-carboxylic acid
pH 7.1, 23°C
0.024
4'-(dimethylamino)-azobenzene-2-carboxylic acid
wild-type, pH 7.1, temperature not specified in the publication
0.0247
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant W105F, pH 7.1, 23°C
0.0251
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant W105H, pH 7.1, 23°C
0.0254
4'-(dimethylamino)-azobenzene-2-carboxylic acid
wild-type, pH 7.1, 23°C
0.027
4'-(dimethylamino)-azobenzene-2-carboxylic acid
cofactor FMN, pH 7.4, 30°C
0.0276
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant W105Y, pH 7.1, 23°C
0.0282
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant W105Q, pH 7.1, 23°C
0.029
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant N121Q, pH 7.1, temperature not specified in the publication
0.031
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant R21K/N121A, pH 7.1, temperature not specified in the publication
0.034
4'-(dimethylamino)-azobenzene-2-carboxylic acid
mutant R21K/N121Q, pH 7.1, temperature not specified in the publication
0.08
NADH
mutant R21G/N121A, pH 7.1, temperature not specified in the publication
0.115
NADH
mutant R21K/N121Q, pH 7.1, temperature not specified in the publication
0.122
NADH
mutant R21K, pH 7.1, temperature not specified in the publication
0.143
NADH
wild-type, pH 7.1, temperature not specified in the publication
0.19
NADH
mutant R21G/N121Q, pH 7.1, temperature not specified in the publication
0.267
NADH
mutant R21K/N121Q, pH 7.1, temperature not specified in the publication
0.371
NADH
mutant N121Q, pH 7.1, temperature not specified in the publication
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
steady-state kinetic analysis
-
additional information
additional information
nonlinear Michaelis-Menten kinetics
-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
181
substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid, pH 7.1, 23°C
188.6
purified recombinant enzyme, pH 7.4, 30°C, methyl red with FMN
25.8
30°C, pH 7.4, substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid
26.6
substrate 4'-(dimethylamino)-azobenzene-2-carboxylic acid, pH 7.4, 30°C
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10 - 50
the enzyme activity drops largely after 50°C, and there is no activity left when temperature rises to 70°C
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
additional information
physiological function
the enzyme catalyses the initial degradation step of azo dyes, characterized by one or more R1-N=N-R2 bonds
physiological function
the enzyme is responsible for the degradation of azo compounds. Residue Arg59 decides the substrate specificity of AzoR
physiological function
-
the enzyme catalyses the initial degradation step of azo dyes, characterized by one or more R1-N=N-R2 bonds
-
additional information
AzoR in its reduced form reveals a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The enzyme performs a conformational change of the isoalloxazine ring and the solvent rearrangement upon reduction
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
42000
48000
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
additional information
?
x * 25000, recombinant His-tagged enzyme, SDS-PAGE, x * 25350, about, His-tagged enzyme, sequence calculation
?
-
x * 25000, recombinant His-tagged enzyme, SDS-PAGE, x * 25350, about, His-tagged enzyme, sequence calculation
-
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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
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 Ã
resolution structure of oxidized AzoR as a search model, modelling
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, 15°C, 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
to 1.6 A resolution, space group F222, with unit-cell parameters a = 72.1, b = 95.5, c = 146.1 A
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
W105A
complete loss of both affinity for FMN and enzyme activity
W105F
31fold decrease in vmax value, Km value similar to wild-type
W105G
complete loss of both affinity for FMN and enzyme activity
W105H
8fold decrease in vmax value, Km value similar to wild-type
W105Q
68fold decrease in vmax value, Km value similar to wild-type
W105A
-
complete loss of both affinity for FMN and enzyme activity
-
W105F
-
31fold decrease in vmax value, Km value similar to wild-type
-
W105H
-
8fold decrease in vmax value, Km value similar to wild-type
-
W105Q
-
68fold decrease in vmax value, Km value similar to wild-type
-
R59A
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
-
mutation influences the formation of dilution-induced intermediates. Mutant R59G contains only two types of FMN, emitting at 530 nm and 600 nm
additional information
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
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
10 - 40
purified recombinant enzyme, pH 7.0, 0.1 M sodium phosphate buffer, 30 min, stable at
65
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
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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
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant His-tagged enzyme from Escherichia coli strain XL1-Blue by nickel affinity chromatography
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
gene acpD, sequence comparisons, recombinant overexpression of the gene provides Escherichia coli strain JM109 with more than 800 times increased azoreductase activity
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)
gene azoR, recombinant expression of His-tagged wild-type and mutant AzoR enzymes in Escherichia coli BL21(DE3)
gene azoR, recombinant overexpression of His-tagged enzyme in Escherichia coli strain XL1-Blue
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ito, K.; Nakanishi, M.; Lee, W.-C.; Sasaki, H.; Zenno,S.; Saigo, K.; Kitade, Y.; Tanokura, M.
Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli
Acta Crystallogr. Sect. F
61
399-402
2005
Escherichia coli (P41407)
-
brenda
Nakanishi, M.; Yatome, C.; Ishida, N.; Kitade, Y.
Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase
J. Biol. Chem.
276
46394-46399
2001
Escherichia coli (P41407)
-
brenda
Ito, K.; Nakanishi, M.; Lee, W.-C.; Zhi, Y.; Sasaki, H.; Zenno, S.; Saigo, K.; Kitade, Y.; Tanokura, M.
Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis
J. Biol. Chem.
283
13889-13896
2008
Escherichia coli (P41407)
-
brenda
Qi, J.; Schloemann, M.; Tischler, D.
Biochemical characterization of an azoreductase from Rhodococcus opacus 1CP possessing methyl red degradation ability
J. Mol. Catal. B
130
9-17
2016
Rhodococcus opacus (A0A1B1KJ01), Rhodococcus opacus 1CP (A0A1B1KJ01)
-
brenda
Mercier, C.; Chalansonnet, V.; Orenga, S.; Gilbert, C.
Characteristics of major Escherichia coli reductases involved in aerobic nitro and azo reduction
J.Appl. Microbiol.
115
1012-1022
2013
Escherichia coli (P41407)
-
brenda
Correia, B.; Chen, Z.; Mendes, S.; Martins, L.; Bento, I.
Crystallization and preliminary X-ray diffraction analysis of the azoreductase PpAzoR from Pseudomonas putida MET94
Acta Crystallogr. Sect. F
67
121-123
2011
Pseudomonas putida (Q88IY3), Pseudomonas putida DSM 6125 (Q88IY3)
brenda
Ooi, T.; Shibata, T.; Sato, R.; Ohno, H.; Kinoshita, S.; Thuoc, T.; Taguchi, S.
An azoreductase, aerobic NADH-dependent flavoprotein discovered from Bacillus sp. Functional expression and enzymatic characterization
Appl. Microbiol. Biotechnol.
75
377-386
2007
Bacillus sp. B29 (Q0WXX2)
brenda
Matsumoto, K.; Mukai, Y.; Ogata, D.; Shozui, F.; Nduko, J.M.; Taguchi, S.; Ooi, T.
Characterization of thermostable FMN-dependent NADH azoreductase from the moderate thermophile Geobacillus stearothermophilus
Appl. Microbiol. Biotechnol.
86
1431-1438
2010
Geobacillus stearothermophilus (Q9X4K2)
brenda
Liu, Z.; Chen, H.; Shaw, N.; Hopper, S.; Chen, L.; Chen, S.; Cerniglia, C.; Wang, B.
Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis
Arch. Biochem. Biophys.
463
68-77
2007
Enterococcus faecalis (Q831B2)
brenda
Sun, J.; Kweon, O.; Jin, J.; He, G.; Li, X.; Cerniglia, C.; Chen, H.
Mutation network-based understanding of pleiotropic and epistatic mutational behavior of Enterococcus faecalis FMN-dependent azoreductase
Biochem. Biophys. Rep.
12
240-244
2017
Enterococcus faecalis (Q831B2), Enterococcus faecalis ATCC 700802 (Q831B2)
brenda
Langer, S.; Nakanishi, S.; Mathes, T.; Knaus, T.; Binter, A.; Macheroux, P.; Mase, T.; Miyakawa, T.; Tanokura, M.; Mack, M.
The flavoenzyme azobenzene reductase AzoR from Escherichia coli binds roseoflavin mononucleotide (RoFMN) with high affinity and is less active in its RoFMN form
Biochemistry
52
4288-4295
2013
Escherichia coli (P41407)
brenda
Misal, S.A.; Lingojwar, D.P.; Lokhande, M.N.; Lokhande, P.D.; Gawai, K.R.
Enzymatic transformation of nitro-aromatic compounds by a flavin-free NADH azoreductase from Lysinibacillus sphaericus
Biotechnol. Lett.
36
127-131
2014
Lysinibacillus sphaericus
brenda
Feng, J.; Han, T.; Zhang, M.; Zhou, Y.; Wu, Q.
Application of 2D fluorescence correlation method to investigate the dilution-induced heterogeneous distribution of the bound FMN in azoreductase
Chin. Chem. Lett.
26
210-214
2015
Escherichia coli
-
brenda
Punj, S.; John, G.
Purification and identification of an FMN-dependent NAD(P)H azoreductase from Enterococcus faecalis
Curr. Issues Mol. Biol.
11
59-66
2009
Enterococcus faecalis, Enterococcus faecalis ATCC 27274
brenda
Ooi, T.; Ogata, D.; Matsumoto, K.; Nakamura, G.; Yu, J.; Yao, M.; Kitamura, M.; Taguchi, S.
Flavin-binding of azoreductase Direct evidences for dual-binding property of apo-azoreductase with FMN and FAD
J. Mol. Catal. B
74
204-208
2012
Bacillus sp. (in: Bacteria) (Q0WXX2), Bacillus sp. (in: Bacteria) B29 (Q0WXX2)
-
brenda
Chen, H.; Xu, H.; Kweon, O.; Chen, S.; Cerniglia, C.
Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis
Microbiology
154
2659-2667
2008
Enterococcus faecalis (Q831B2), Enterococcus faecalis ATCC 700802 (Q831B2)
brenda
Chen, H.; Wang, R.; Cerniglia, C.
Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis
Protein Expr. Purif.
34
302-310
2004
Enterococcus faecalis (Q831B2), Enterococcus faecalis ATCC 19433 (Q831B2)
brenda
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