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1.7.1.17: FMN-dependent NADH-azoreductase

This is an abbreviated version!
For detailed information about FMN-dependent NADH-azoreductase, go to the full flat file.

Reaction

anthranilate
+
N,N-dimethyl-1,4-phenylenediamine
+ 2 NAD+ =
2-(4-dimethylaminophenyl)diazenylbenzoate
+ 2 NADH + 2 H+

Synonyms

acpD, AZO, azo-dye reductase, AzoA, AzoR, AzoR1, azoreductase, AzoRo, AzrA, AzrG, flavin-containing oxygen-insensitive azoreductase, FMN dependent azoreductase, FMN-dependent methyl red reductase, FMN-dependent NADH-azo compound oxidoreductase, FMN-dependent NADH-azo reductase, FMN-dependent NADH-azoreductase, FMN-dependent-NADH azoreductase, indigo reductase, NADH-azoreductase

ECTree

     1 Oxidoreductases
         1.7 Acting on other nitrogenous compounds as donors
             1.7.1 With NAD+ or NADP+ as acceptor
                1.7.1.17 FMN-dependent NADH-azoreductase

Crystallization

Crystallization on EC 1.7.1.17 - FMN-dependent NADH-azoreductase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with FMN, sitting drop vapor diffusion method, using 0.17 M ammonium sulfate and 25.2% (w/v) PEG 4000
purified recombinant His-tagged wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and Y151F enzyme mutant in complex with FMN, sitting drop vapor diffusion method, mixing of 0.002 ml of 42 mg/ml protein solution with an equal volume of reservoir solution containing 40% PEG 600 and 100 mM CHES, pH 9.5 for the wild-type, and 36% PEG 600 and 100 mM phosphate-citrate buffer, pH 4.2 for the mutant, equilibration against 0.1 m reservoir resolution, 20°C, X-ray diffraction structure determination and analysis at 1.97 and 1.95 A resolution respectively, molecular replacement method. Diffraction-quality crystals for the Y127F mutant cannot be obtained
docking simulations. The isoalloxazine ring of FAD localizes at the same site and plays the same role as that of FMN in AzrA
in complex with FMN, to 2.07 A resolution. The AzoA monomer shows a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind
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
to 1.6 A resolution, space group F222, with unit-cell parameters a = 72.1, b = 95.5, c = 146.1 A