Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).
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PMID:A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties. 1 Dec 14

Nitroalkane compounds are widely used in chemical industry and are also produced by microorganisms and plants. Some nitroalkanes have been demonstrated to be carcinogenic, and enzymatic oxidation of nitroalkanes is of considerable interest. 2-Nitropropane dioxygenases from Neurospora crassa and Williopsis mrakii (Hansenula mrakii), members of one family of the nitroalkane-oxidizing enzymes, contain FMN and FAD, respectively. The enzymatic oxidation of nitroalkanes by 2-nitropropane dioxygenase operates by an oxidase-style catalytic mechanism, which was recently shown to involve the formation of an anionic flavin semiquinone. This represents a unique case in which an anionic flavin semiquinone has been experimentally observed in the catalytic pathway for oxidation catalyzed by a flavin-dependent enzyme. Here we report the first crystal structure of 2-nitropropane dioxygenase from Pseudomonas aeruginosa in two forms: a binary complex with FMN and a ternary complex with both FMN and 2-nitropropane. The structure identifies His(152) as the proposed catalytic base, thus providing a structural framework for a better understanding of the catalytic mechanism.
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PMID:Crystal structure of 2-nitropropane dioxygenase complexed with FMN and substrate. Identification of the catalytic base. 1668 7

2-Nitropropane dioxygenase from Hansenula mrakii was expressed in Escherichia coli cells and purified in active and stable form using 60% saturation of ammonium sulfate and a single chromatographic step onto a DEAE column. MALDI-TOF mass spectrometric and spectrophotometric analyses of the flavin extracted by heat or acid denaturation of the enzyme indicated that FMN, and not FAD as erroneously reported previously, is present in a 1:1 stoichiometry with the protein. Inductively coupled plasma mass spectrometric analysis of the enzyme established that H. mrakii 2-nitropropane dioxygenase contains negligible amounts of iron, manganese, zinc, and copper ions, which are not catalytically relevant. Anaerobic substrate reduction and kinetic data using a Clark oxygen electrode to measure rates of oxygen consumption indicated that the enzyme is active on a broad range of alkyl nitronates, with a marked preference for unbranched substrates over propyl-2-nitronate. Interestingly, the enzyme reacts poorly, if at all, with nitroalkanes, as suggested by lack of both anaerobic reduction of the enzyme-bound flavin and consumption of oxygen with nitroethane, nitrobutane, and 2-nitropropane. Finally, both the tight binding of sulfite (K(d)=90 microM, at pH 8 and 15 degrees C) to the enzyme and the formation of the anionic flavosemiquinone upon anaerobic incubation with alkyl nitronates are consistent with the presence of a positively charged group in proximity of the N1-C2=O atoms of the FMN cofactor.
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PMID:Oxidation of alkyl nitronates catalyzed by 2-nitropropane dioxygenase from Hansenula mrakii. 1832 75