Literature summary for 1.7.1.17 extracted from

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 (2008), J. Biol. Chem., 283, 13889-13896 .

No PubMed abstract available

Cloned(Commentary)

Cloned (Comment)	Organism
gene azoR, recombinant expression of His-tagged wild-type and mutant AzoR enzymes in Escherichia coli BL21(DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Escherichia coli

Crystallization (Comment)

Organism

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

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
F162A	site-directed mutagenesis	Escherichia coli
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	Escherichia coli
Y120A	site-directed mutagenesis	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
dicoumarol	enzyme-bound structure analysis, overview	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten kinetics	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+	Escherichia coli	i.e. azo dye methyl red	anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P41407	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant AzoR enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography	Escherichia coli

Reaction

Reaction	Comment	Organism	Reaction ID
anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+ = 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+	ping-pong reaction mechanism, overview	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2-(4-dimethylaminophenyl)diazenylbenzoate + 2 NADH + 2 H+	i.e. azo dye methyl red	Escherichia coli	anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD+	-	?
additional information	active site structure analysis and substrate specificity, Azo compound binding structure, overview	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
AzoR	-	Escherichia coli
azoreductase	-	Escherichia coli
FMN-dependent NADH-azoreductase	-	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
FMN	enzyme-bound structure analysis, overview	Escherichia coli
NADH	-	Escherichia coli

General Information

General Information	Comment	Organism
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	Escherichia coli
physiological function	the enzyme is responsible for the degradation of azo compounds. Residue Arg59 decides the substrate specificity of AzoR	Escherichia coli