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Information on EC 1.7.1.6 - azobenzene reductase and Organism(s) Pseudomonas aeruginosa and UniProt Accession Q9I5F3

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IUBMB 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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This record set is specific for:
Pseudomonas aeruginosa
UNIPROT: Q9I5F3
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The taxonomic range for the selected organisms is: Pseudomonas aeruginosa
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
azo reductase, azo-reductase, paazor1, orange ii azoreductase, azobenzene reductase, azor1, ef0404, aerobic azoreductase, orange i azoreductase, azo 1, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
class 1 azoreductase
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azo dye reductase
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azo reductase
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azo-dye reductase
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azoreductase
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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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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N,N-dimethyl-1,4-phenylenediamine + aniline + 2 NADP+ = 4-(dimethylamino)azobenzene + 2 NADPH + 2 H+
show the reaction diagram
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
N,N-dimethyl-1,4-phenylenediamine + aniline + 2 NADP+ = 4-(dimethylamino)azobenzene + 2 NADPH + 2 H+
show the reaction diagram
ping-pong mechanism
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
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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 hide
9029-31-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-(4-aminophenylazo)benzenesulfonic acid + NADPH + H+
?
show the reaction diagram
about 25% specific activity compared to balsalazide
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-
?
balsalazide + NADPH + H+
?
show the reaction diagram
100% specific activity
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-
?
balsalazide + NADPH + H+
? + NADP+
show the reaction diagram
tautomeric forms of balsalazide occur in solution, reaction mechanism, overview
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-
?
methyl red + NADH + H+
?
show the reaction diagram
the enzyme exhibits lower activity with NADH compared to NADPH
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-
?
methyl red + NADPH + H+
?
show the reaction diagram
about 50% specific activity compared to balsalazide
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-
?
olsalazine + NADPH + H+
?
show the reaction diagram
less than 10% specific activity compared to balsalazide
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-
?
sulfasalazine + NADPH + H+
?
show the reaction diagram
about 20% specific activity compared to balsalazide
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?
Amaranth + NADH
?
show the reaction diagram
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-
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?
Amido black + NADH
?
show the reaction diagram
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?
Atul acid black + NADH
?
show the reaction diagram
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?
balsalzide + NADH + H+
? + NAD+
show the reaction diagram
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?
Eriochrome blue black + NADH
?
show the reaction diagram
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?
methyl red + NADPH + H+
? + NADP+
show the reaction diagram
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?
Navitan fast blue S5R + NADH
metanilic acid + 1,4-diamino naphtalene + peri acid + NAD+
show the reaction diagram
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?
Orange G + NADH
?
show the reaction diagram
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?
sulfasalazine + NADPH + H+
? + NADP+
show the reaction diagram
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?
Tropaeolin + NADH
?
show the reaction diagram
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
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isoform AzoR1 prefers NADPH over NADH
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cibacron blue
a competitive NAD(P)H inhibitor
Fe2+
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complete inhibition
Methyl red
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substrate inhibition above 0.08 mM
NaCl
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more than 40% of maximum activity up to 2.5 M
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0986 - 0.1029
balsalazide
0.0445 - 0.0927
Methyl red
0.538 - 0.965
NADH
0.535 - 1.197
NADPH
0.055 - 2.447
balsalzide
0.26 - 0.983
Methyl red
0.298 - 4.35
NADH
10.26
NADPH
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isoform AzoR1, pH 8.0, temperature not specified in the publication
0.063
Navitan fast blue S5R
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pH 7.0, room temperature
0.22 - 1.118
sulfasalazine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
24.1 - 36.8
balsalazide
12.7 - 19.1
Methyl red
10 - 25.2
NADH
33.6 - 35.5
NADPH
0.2 - 20
balsalzide
1 - 11.6
Methyl red
0.5 - 2.6
sulfasalazine
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
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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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
phylogeny of azoreductases, overview
physiological function
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29000
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SDS-PAGE, native PAGE, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 29000, subunit mass by SDS-PAGE, native mass by gel filtration
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
mutant enzyme Y131F in the presence of methyl red, sitting drop vapor diffusion method, using 0.1 M sodium acetate pH 4.5, 1.0 M diammonium hydrogen phosphate
structure of balsalazide bound to paAzoR1, PDBs ID 3LT5
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y131F
the mutant shows increased specific activity with methyl red and reduced specific activity with balsalazide compared to the wild type enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42 - 55
whilst wild type AzoR1 loses 50% of its activity after incubation for 10 min at 55°C, mutant enzyme Y131F loses 50% of its activity after exposure to 42°C for 10 min, both enzymes are inactive after 10 min at 80°C
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) pLysS cells
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
in presence of balsalazide, expression of isoform AzoR1 increases by about 50%
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in presence of methyl red or balsalzide, expression of isoform AzoR2 increases 3.5- and 2.5fold, respectively
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in presence of methyl red, expression of isoform AzoR1 increases more than 50%
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
azoreductases from enteric bacteria are targets in the development of drugs for the treatment of colon related disorders
environmental protection
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potential for the treatment of azo dye contaminated wastewater
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nachiyar, C.V.; Rajakumar, G.S.
Purification and characterization of an oxygen insensitive azoreductase from Pseudomonas aeruginosa
Enzyme Microb. Technol.
36
503-509
2005
Pseudomonas aeruginosa
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Manually annotated by BRENDA team
Wang, C.; Laurieri, N.; Abuhammad, A.; Lowe, E.; Westwood, I.; Ryan, A.; Sim, E.
Role of tyrosine 131 in the active site of paAzoR1, an azoreductase with specificity for the inflammatory bowel disease prodrug balsalazide
Acta Crystallogr. Sect. F
66
2-7
2009
Pseudomonas aeruginosa (Q9I5F3), Pseudomonas aeruginosa
Manually annotated by BRENDA team
Ryan, A.; Wang, C.; Laurieri, N.; Westwood, I.; Sim, E.
Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases
Protein Cell
1
780-790
2010
Pseudomonas aeruginosa
Manually annotated by BRENDA team
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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Manually annotated by BRENDA team