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Information on EC 1.7.1.6 - azobenzene reductase and Organism(s) Enterococcus faecalis and UniProt Accession Q831B2
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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
-
decolor
-
textile
- laccase
-
veratryl
- environmental protection
-
remazol
- drug development
-
dechlorinase
- dcip
-
rubine
- 2,6-dinitrotoluene
-
ponceau
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voltammograms
- industry
- laterosporus
- dehydrochlorinase
- degradation
- analysis
The taxonomic range for the selected organisms is: Enterococcus faecalis
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, azor1, ef0404, azobenzene reductase, aerobic azoreductase, orange i azoreductase, azoreductase 1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
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
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
reduction
redox reaction
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-
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oxidation
reduction
oxidation
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reduction
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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
1-(4'-carboxyphenylazo)-2-naphthol + NADH
4-amino-1-benzoic acid + NAD+ + 1-amino-2-hydroxynaphthalene
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substrate is carboxy-Orange II
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-
?
2-[4-(dimethylamino)phenylazo]benzoic acid + NADH
2-aminobenzoic acid + N,N-dimethyl-1,4-phenylenediamine + NAD+
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substrate is Methyl red
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-
?
4-dimethylaminoazobenzene-2'-carboxylic acid + 2 NADPH + 2 H+
anthranilate + N,N'-dimethylamino-aniline + 2 NADP+
i.e. methyl red
-
-
?
methyl red + 2 NADH + 2 H+
N,N-dimethyl-p-phenylenediamine + 2-aminobenzoic acid + 2 NAD+
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?
additional information
?
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important for the conversion of azo dyes in the gastrointestinal tract
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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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?
additional information
?
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the enzyme can also catalyze the nitroreduction of 7-nitrocoumarin-3-carboxylic acid in a FMN-dependent manner
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?
additional information
?
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the enzyme can also catalyze the nitroreduction of 7-nitrocoumarin-3-carboxylic acid in a FMN-dependent manner
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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
additional information
?
-
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important for the conversion of azo dyes in the gastrointestinal tract
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADPH
180fold preference for NADH over NADPH as an electron donor to reduce methyl red
NADH
180fold preference for NADH over NADPH as an electron donor to reduce methyl red
FMN
enzyme-bound prosthetic group, FMN-dependence for azoreductase activity
NADH
-
AzoA shows more than 180fold preference for NADH over NADPH as an electron donor to reduce methyl red
NADPH
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AzoA shows more than 180fold preference for NADH over NADPH as an electron donor to reduce methyl red
additional information
the enzyme can use both cofactors, NADH and NADPH, it shows a preference towards NADH for the azoreductase activity, but a preference towards NADPH for the nitroreductase activity
-
additional information
-
the enzyme can use both cofactors, NADH and NADPH, it shows a preference towards NADH for the azoreductase activity, but a preference towards NADPH for the nitroreductase activity
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.003
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crude extract, using methyl red and NADPH as substrates, in 50 mM potassium phosphate buffer pH 7.0 at 25°C
626
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after 209fold purification, using methyl red and NADPH as substrates, in 50 mM potassium phosphate buffer pH 7.0 at 25°C
63.9
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purified enzyme, pH 7.1, 23°C, with Methyl red and NADH as substrates
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
-
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
evolution
physiological function
evolution
enzyme EF0404 shows both nitroreductase and azoreductase activity. The biochemical characteristics, such as substrate and cofactor specificity, of enzyme EF0404 resemble the properties of the known azoreductase AzoA. But its sequence matches with the nitroreductase group B, the same as enzyme EF0648
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
physiological function
enzyme EF0404 shows both nitroreductase and azoreductase activity. The biochemical characteristics, such as substrate and cofactor specificity, of enzyme EF0404 resemble the properties of the known azoreductase AzoA. But its sequence matches with the nitroreductase group, the same as enzyme EF0648
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
23000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
modeling of structure. Binding mode shows that the benzoic acid moiety of substrate methyl red and the nicotinamide ring of NADH are not parallel to the flavin isoalloxazine ring, but lay against it at angles of about 45 and 35 degrees, respectively. The adenine ribose moiety of NADH is surrounded by loop l2 on chain B and alpha3 on chain A in a typical Rossmann fold
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
W105A
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mutant, complete loss of both affinity for FMN and enzyme activity
W105G
-
mutant, complete loss of both affinity for FMN and enzyme activity
W105Y
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mutant, substitution does not significantly decrease the Vmax of the enzyme, 22% reduction
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
cell extracts of Enterococcus faecalis are prepared, AzoA is purified by hydrophobic, anion exchange and affinity chromatography
octyl Sepharose column chromatography, DEAE Bio-gel agarose column chromatography, and Affi-gel Blue gel filtration
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recombinant FMN cofactor-bound His-tagged enzyme from Escherichia coli strain XL1-Blue by affinity chromatography and dialysis
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene EF_0404, phylogenetic analysis and tree, recombinant overexpression of His-tagged enzyme in Escherichia coli strain XL1-Blue from vector pQE30
into the vector pET-11a for expression in Escherichia coli BL21-Gold DE3 pLysS cells
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
degradation
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expression of enzyme gene AzoA in Escherichia coli induces a higher rate of dye reduction with increases of 2fold for methyl red, 4fold for ponceau BS and 2.6fold for orange II compared to noninduced cells, respectively
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
Chen, H.; Wang, R.F.; Cerniglia, C.E.
Molecular cloning, overexpression, purification, and characterization of an aerobic FMN-dependent azoreductase from Enterococcus faecalis
Protein Expr. Purif.
34
302-310
2004
Enterococcus faecalis
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
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)
Punj, S.; John, G.H.
Purification and identification of an FMN-dependent NAD(P)H azoreductase from Enterococcus faecalis
Curr. Issues Mol. Biol.
11
59-65
2009
Enterococcus faecalis (Q831B2), Enterococcus faecalis
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
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
Feng, J.; Kweon, O.; Xu, H.; Cerniglia, C.E.; Chen, H.
Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis
Arch. Biochem. Biophys.
520
99-107
2012
Enterococcus faecalis (Q831B2)
Feng, J.; Heinze, T.; Xu, H.; Cerniglia, C.; Chen, H.
Evidence for significantly enhancing reduction of azo dyes in Escherichia coli by expressed cytoplasmic azoreductase (AzoA) of Enterococcus faecalis
Protein Pept. Lett.
17
578-584
2010
Enterococcus faecalis
Chalansonnet, V.; Mercier, C.; Orenga, S.; Gilbert, C.
Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
BMC Microbiol.
17
126
2017
Enterococcus faecalis (Q838N5), Enterococcus faecalis
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
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