the 3HAO reaction is initiated by substrate binding, which induces closure of the active site. Oxygen then binds to the active-site iron. Transfer of an electron from the active-site iron to oxygen creates an oxygen radical, thereby facilitating the addition of both O atoms to 3-HANA and forming 2-amino-3-carboxymuconic 6-semialdehyde (ACMS), an active intermediate. ACMS then spontaneously cyclizes to form quinolinic acid
each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A
it is possible that inhibition of 3-HAD may improve neurologic status through an increased production of kynurenic acid, a non-specific inhibitor of excitatory amino acid receptors and an inhibitor of quinolinic acid neurotoxicity
the inactivation results in the consumption of 2 equivalents of oxygen and the production of superoxide. The inhibitor stimulates the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and FeI(II). The nhibitor does not form an adduct with the enzyme. Four conserved cysteines are oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site
Changes in mouse liver and chicken embryo yolk sac membrane soluble proteins due to an organophosphorous insecticide (OPI) diazinon linked to several noncholinergic OPI effects in mice and chicken embryos.
enzyme 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2 to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid. Quinolinic acid is a highly potent excitotoxin that is implicated in a number of neurodegenerative conditions
oxidative CC bond cleavage of 2-amino-4-tert-butylphenolate on complex nonheme iron(II) complex, [(6-Me3-TPA)FeII(4-tBu-HAP)](ClO4) is mimicking the enzyme function. The iron(II)-aminophenolate complex reacts with molecular oxygen in acetonitrile under ambient conditions, overview
enzyme molecular modeling, the water population of the active site, and the protein flexibility as well as the amino acid residues interaction networks are relevant for the enzyme activity, hybrid quantum-mechanics/molecular-mechanics (QM/MM) calculations
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first vapor diffusion with sitting drop method starting from protein concentration of 10 mg/ml in 32% (NH4)2SO4, 0.1 M sodium acetate, 10 mM 2-mercaptoethanol, pH 5, subsequently, crystals can be obtained by seeding starting from fragments of first crystallization experiments using 40% (NH4)2SO4, Tris-HCl, 40 mM MgCl2, 3% MPD, pH 8 as precipitant
purified recombinant detagged enzyme in complex with Zn2+ or Fe2+, protein with zinc sulfate or iron sulfate best from 0.1 M HEPES, pH 7.5, 2% PEG 400, and 2.0 M ammonium sulfate., in 2-7 days, X-ray diffraction structure determination and analysis at 1.75-1.88 A resolution, modeling
bovine kidney homogenized in 0.1 M potassium dihydrogen phosphate buffer with 20% glycerol and 3 mM 2-mercaptoethanol, pH 7.4, centrifuged, protein fraction of supernatant that precipitates from 34-62% saturated (NH4)2SO4 is resuspended in 5 mM potassium dihydrogen phosphate buffer with 20% glycerol and 3 mM 2-mercaptoethanol, pH 7.4, dialyzed against same buffer containing protease inhibitor PMSF (0.1 mM), applied to DEAE Sephadex A-50 column, fractions with enzyme activity pooled and concentrated by precipitation with 80%-saturated (NH4)2SO4, dialyzed against 10 mM Tris-HCl with 20% glycerol and 3 mM 2-mercaptoethanol, pH 7.4, applied to Blue-Sepharose CL-4B column, concentration of active fractions by ultrafiltration through YM-10 membrane
recombinant His6-MBP-tagged enzyme from Escherichia coli by nickel affinity chromatography and cleavage of the His-tag by TEV protease, elimination of the tags by nickel affinity and amylose affinity chromatography, te final step is gel filtration
Y3HAO protein is subcloned into the pET-28a expression vector from extracted Saccharomyces cerevisiae genomic DNA, and the Y3HAO protein is highly expressed as a soluble protein in Escherichia coli strain BL21(DE3) with a six-residueHis tag attached to its N-terminus
Muraki, T.; Taki, M.; Hasegawa, Y.; Iwaki, H.; Lau, P.C.
Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomonas fluorescens strain KU-7