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Information on EC 1.7.1.6 - azobenzene reductase and Organism(s) Escherichia coli and UniProt Accession P41407
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1.7.1.6 azobenzene reductaseIUBMB Comments
The reaction occurs in the reverse direction to that shown above. Other azo dyes, such as Methyl Red, Rocceline, Solar Orange and Sumifix Black B can also be reduced .
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Word Map
- 1.7.1.6
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decolor
-
textile
- laccase
-
veratryl
- environmental protection
-
remazol
- drug development
-
dechlorinase
- dcip
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rubine
- 2,6-dinitrotoluene
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ponceau
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voltammograms
- industry
- laterosporus
- dehydrochlorinase
- degradation
- analysis
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
Synonyms
azo reductase, azo-reductase, paazor1, orange ii azoreductase, azobenzene reductase, azor1, ef0404, aerobic azoreductase, orange i azoreductase, azo 1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
AzoR
azo dye reductase
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azo reductase
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azo-dye reductase
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azoreductase
dibromopropylaminophenylazobenzoic azoreductase
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dimethylaminobenzene reductase
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methyl red azoreductase
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N,N-dimethyl-4-phenylazoaniline azoreductase
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NAD(P)H:1-(4'-sulfophenylazo)-2-naphthol oxidoreductase
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NADPH2-dependent azoreductase
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NC-reductase
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new Coccine (NC)-reductase
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nicotinamide adenine dinucleotide (phosphate) azoreductase
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Orange II azoreductase
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p-aminoazobenzene reductase
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p-dimethylaminoazobenzene azoreductase
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reductase, azobenzene
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azoreductase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
N,N-dimethyl-1,4-phenylenediamine + aniline + 2 NADP+ = 4-(dimethylamino)azobenzene + 2 NADPH + 2 H+
the catalytic reaction requires tautomerisation of the azo compound to a quinoneimine and provides a unifying mechanism for the reduction of azo substrates by azoreductases, mechanism for azoreduction by FMN-dependent azoreductases, overview. N5 of FMN accepts a hydride during oxidation of NAD(P)H and donates it upon reduction of the substrate
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
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oxidation
reduction
oxidation
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reduction
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reduction
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catalyzes the reductive cleavage of azo groups by a ping-pong mechanism
SYSTEMATIC NAME
IUBMB Comments
N,N-dimethyl-1,4-phenylenediamine, aniline:NADP+ oxidoreductase
The reaction occurs in the reverse direction to that shown above. Other azo dyes, such as Methyl Red, Rocceline, Solar Orange and Sumifix Black B can also be reduced [2].
CAS REGISTRY NUMBER
COMMENTARY
9029-31-6
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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
2-methyl-1,4-naphthoquinone + NADH + H+
?
menadione, vitamin K3
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?
4'-diethylaminoazobenzene-2-carboxylic acid + NADH + H+
?
ethyl red
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?
4'-dimethylaminoazobenzene-2-carboxylic acid + NADH + H+
2-aminobenzoic acid + N,N'-dimethyl-p-phenylenediamine + NAD+
methyl red
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?
4-dimethylaminoazobenzene-2'-carboxylic acid + 2 NADH + 2 H+
anthranilate + N,N'-dimethylaminoaniline + 2 NAD+
i.e. methyl red
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-
?
1-(4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid + NADPH
alpha-naphthylamine-4-sulfonic acid + beta-naphthol-3,6-disulfonic acid + NADP+
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?
1-methyl-1-([1-[(Z)-(methyl[4-[(E)-2-[6'-[(E)-[4-[methyl([4-[(1-methylpyrrolidinium-1-yl)methyl]-1H-1,2,3-triazol-1-yl]methyl)amino]phenyl]diazenyl]-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthen]-3'-yl]ethenyl]phenyl]iminio)methyl]-1H-1,2,3-triazol-4-yl]methyl)pyrrolidinium + FMN + NADH + H+
? + NAD+
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pro-fluorophore, based on fluorescent rhodamine 110. Upon reduction of the two diazo bonds, substrate shows a green fluorescence suitable for the detection of azoreductases
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-
?
4-[(2-hydroxy-1-naphthalenyl)azo]benzene sulfonic acid + NAD(P)H
4-amino-1-benzenesulfonic acid + NAD(P)+ + 1-amino-2-hydroxynaphthalene
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NADH is more effective
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?
methyl red + NADH
2-aminobenzoic acid + N,N-dimethyl-1,4-phenylenediamine + NAD+
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?
additional information
?
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enzyme additionally shows quinone reductase activity. When NAD(P)H is used as an electron donor, the purified enzyme can reduce menadione effectively with a quinone reductase activity of approximately 3.4 U ml-1
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?
additional information
?
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in general, the substrates of azoreductases do not make many specific hydrophilic interactions, which explains the ability of the active site to accommodate a range of hydrophobic substrates
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
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the flavin mononucleotide analogue 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. The AzoR-roseoflavin mononucleotide holoenzyme complex shows 30% of AzoR-FMN activity in a standard assay
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FMN
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a flavin-dependent azoreductase, FMN is anchored by a series of sequence-independent hydrogen bonds to a structural motif referred to as the FMN binding cradle. One molecule of flavin is bound within each active site and is required for activity
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
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changes in salinity from 0 to 2% do not affect the activity of the enzyme very much. After a 1-h incubation, more than 80% relative activity is indicated at 0-5% salt concentrations
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-methylhydroquinone
0.5 mM, increase in expression of azoR mRNA relative to the expression at zero time before stress is applied, at 10 min 46.0, at 20 min 129.1
catechol
6 mM, increase in expression of azoR mRNA relative to the expression at zero time before stress is applied, at 10 min 15.6, at 20 min 31.9
Diamide
1 mM, increase in expression of azoR mRNA relative to the expression at zero time before stress is applied, at 10 min 11.4, at 20 min 8.1
H2O2
1 mM, increase in expression of azoR mRNA relative to the expression at zero time before stress is applied, at 10 min 0.9, at 20 min 0.6, 10 mM, 10 min 3.0, 20 min 1.9
menadione
0.3 mM, increase in expression of azoR mRNA relative to the expression at zero time before stress is applied, at 10 min 25.5, at 20 min 13.3
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
45
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
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azoreductases are primarily cytosolic enzymes, but have been shown to be secreted during exposure of bacteria to azo dyes
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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bacterial azoreductases are associated with the activation of two classes of drug, azo drugs for the treatment of inflammatory bowel disease and nitrofuran antibiotics, mechanism of reduction of azo compounds, overview
additional information
additional information
interaction analysis and molecular docking of substrates and enzyme for analysis of binding mode and binding stability between azoreductase and azo dyes, overview
additional information
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active site structure of ecAzoR, overview. The active sites of the enzyme are situated at the dimer interface and are formed by residues from both monomers. One molecule of flavin is bound within each active site and is required for activity
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
12000
28000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
homodimer
homodimer
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homodimeric flavodoxin fold of ecAzoR. The active sites of the enzyme are situated at the dimer interface and are formed by residues from both monomers. One molecule of flavin is bound within each active site and is required for activity
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
the structures of oxidized and reduced AzoR, and in complex with the inhibitor 3,3'-methylene-bis(4-hydroxycoumarin) are determined at resolutions from 1.4 to 2.3 A
hanging drop vapour diffusion method using 10 mM Tris-HCl (pH 8.0), 1 mM FMN, and an equal volume of reservoir solution containing 200 mM NaCl, 100 mM CAPS2 (pH 10.5), 20% (w/v) polyethylene glycol 8000, 20% (v/v) 1,4-dioxane, and 4 mM menadione
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
F162A
Phe-162 is chosen because it is predicted to participate in the substrate binding on top of the isoalloxazine ring, as observed in the AzoR-inhibitor structure
R59A
the results indicate that Arg-59 decides the substrate specificity of AzoR
R59G
site-directed mutagenesis, the interaction of FMN with substrates methyl red and methyl orange changes from van der Waals interaction in the wild-type to hydrogen bonding in the mutant, the association constants decrease
Y120A
Tyr-120 is chosen because it is predicted to participate in the substrate binding on top of the isoalloxazine ring, as observed in the AzoR-inhibitor structure
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
His-tagged proteins are purified by gravity-flow chromatography using nickel-nitrilotriacetic acid-agarose
the GST-AzoR protein is purified by glutathione Sepharose 4B, the GST tag is removed by digestion with thrombin
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
into the vector pET-22b for expression in Escherichia coli BL21DE3 cells
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
industry
industrial use of the enzyme in the biodegradation process of azo compounds
analysis
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pro-fluorophore 1-methyl-1-([1-[(Z)-(methyl[4-[(E)-2-[6'-[(E)-[4-[methyl([4-[(1-methylpyrrolidinium-1-yl)methyl]-1H-1,2,3-triazol-1-yl]methyl)amino]phenyl]diazenyl]-3-oxo-3H-spiro[2-benzofuran-1,9'-xanthen]-3'-yl]ethenyl]phenyl]iminio)methyl]-1H-1,2,3-triazol-4-yl]methyl)pyrrolidinium shows a green fluorescence upon reduction of the two diazo bonds suitable for the detection of azoreductases
drug development
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azoreductases from enteric bacteria are targets in the development of drugs for the treatment of colon related disorders
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ghosh, D.K.; Ghosh, S.; Sadhukhan, P.; Mandal, A.; Chaudhuri, J.
Purification of two azoreductases from Escherichia coli K12
Indian J. Exp. Biol.
31
951-954
1993
Escherichia coli
Chen, H.
Recent advances in azo dye degrading enzyme research
Curr. Protein Pept. Sci.
7
101-111
2006
Acidaminococcus fermentans, Bacillus sp. (in: Bacteria), Bacillus sp. (in: Bacteria) OY-2, Bacillus sp. (in: Bacteria) SF, Bacillus subtilis, Bacteroides thetaiotaomicron, Bifidobacterium adolescentis, Blautia obeum, Cereibacter sphaeroides, Citrobacter sp., Clostridium perfringens, Coprococcus catus, Enterocloster clostridioformis, Enterococcus faecalis, Escherichia coli, Fusobacterium sp., Holdemanella biformis, Kocuria rosea, Kocuria varians, Micrococcus luteus, Pantoea agglomerans, Pigmentiphaga kullae, Pigmentiphaga kullae K24, Proteus vulgaris, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Xenophilus azovorans, Xenophilus azovorans KF46
Ito, K.; Nakanishi, M.; Lee, W.; Sasaki, H.; Zenno, S.; Saigo, K.; Kitade, Y.; Tanokura, M.
Three-dimensional structure of AzoR from Escherichia coli. An oxidoreductase conserved in microorganisms
J. Biol. Chem.
281
20567-20576
2006
Escherichia coli
Bafana, A.; Chakrabarti, T.
Lateral gene transfer in phylogeny of azoreductase enzyme
Comput. Biol. Chem.
32
191-197
2008
Bacillus sp. (in: Bacteria) (Q9FAW5), Bacillus sp. (in: Bacteria) OY1-2 (Q9FAW5), Bacillus subtilis (O07529), Bacillus subtilis 168 (O07529), Cereibacter sphaeroides (Q8GKS3), Cereibacter sphaeroides AS1.1737 (Q8GKS3), Enterococcus faecalis (Q831B2), Escherichia coli (Q8X9S9), Geobacillus stearothermophilus (Q8RR37), Pigmentiphaga kullae (Q6YAN1), Pigmentiphaga kullae K24 (Q6YAN1), Staphylococcus aureus (Q50H63), Xenophilus azovorans (Q8KU07), Xenophilus azovorans KF46F / DSM 13620 (Q8KU07)
Liu, G.; Zhou, J.; Fu, Q.S.; Wang, J.
The Escherichia coli azoreductase AzoR Is involved in resistance to thiol-specific stress caused by electrophilic quinones
J. Bacteriol.
191
6394-6400
2009
Escherichia coli (P41407)
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)
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
Chevalier, A.; Mercier, C.; Saurel, L.; Orenga, S.; Renard, P.; Romieu, A.
The first latent green fluorophores for the detection of azoreductase activity in bacterial cultures
Chem. Commun. (Camb. )
49
8815-8817
2013
Escherichia coli
Cui, D.; Li, G.; Zhao, D.; Gu, X.; Wang, C.; Zhao, M.
Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity
Process Biochem.
47
544-549
2012
Escherichia coli, Escherichia coli CD-2
-
Ryan, A.
Azoreductases in drug metabolism
Br. J. Pharmacol.
174
2161-2173
2017
Bacillus sp. B29 (C0STY1), Bacillus subtilis, Cereibacter sphaeroides, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa (Q9I5F3), Pseudomonas putida
-
Feng, J.; Wu, Q.; Zhou, Y.; Hu, Q.; Fang, R.; Tang, L.
Interaction between flavin mononucleotide-containing azoreductase and azo dyes
Spectrosc. Lett.
49
626-634
2016
Escherichia coli (P41407)
-
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