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Information on EC 1.7.1.17 - FMN-dependent NADH-azoreductase and Organism(s) Escherichia coli and UniProt Accession P41407
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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 taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
fmn-dependent nadh-azoreductase, indigo reductase, azoro, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
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+
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
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.
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-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+
4'-(dimethylamino)-azobenzene-2-carboxylic acid + 2 NADH + 2 H+
2-aminobenzoic acid + N,N-dimethyl-p-phenylenediamine + 2 NAD+
i.e. methyl red
-
-
?
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
-
-
-
?
ethyl red + 2 NADH + 2 H+
anthranilate + N,N-diethyl-1,4-phenylenediamine + 2 NAD+
-
-
-
?
amaranth + 2 NADH + 2 H+
4-aminonaphthalene-1-sulfonic acid + 4-amino-3-hydroxynaphthalene-2,7-disulfonic acid + 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
-
-
?
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
-
-
?
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
-
-
?
7-nitrocoumarin-3-carboxylic acid + NADH + H+
7-aminocoumarin-3-carboxylic acid + NAD+ + H2O
-
-
-
?
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
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
-
adding FMN as cofactor significantly improves the azodye-reducing activity of the cytosolic extract
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
-
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
0.027
4'-(dimethylamino)-azobenzene-2-carboxylic acid
cofactor FMN, pH 7.4, 30°C
additional information
additional information
Michaelis-Menten kinetics
-
additional information
additional information
steady-state kinetic analysis
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
the enzyme is responsible for the degradation of azo compounds. Residue Arg59 decides the substrate specificity of AzoR
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
42000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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 Å 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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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 XL1-Blue by nickel affinity chromatography
recombinant His-tagged wild-type and mutant AzoR enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene acpD, sequence comparisons, recombinant overexpression of the gene provides Escherichia coli strain JM109 with more than 800 times increased azoreductase activity
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
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)
-
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)
-
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)
-
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)
-
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)
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
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