Oxidation of alkyl nitronates catalyzed by 2-nitropropane dioxygenase from Hansenula mrakii.
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.
Mijatovic
Slavica
S
Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA.
Gadda
Giovanni
G
D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology; D009492:Neurospora crassa / Q000201:enzymology; D009595:Nitroparaffins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004718:Saccharomycetales / Q000201:enzymology*; D013379:Substrate Specificity
D003067:Coenzymes; D005656:Fungal Proteins; D009595:Nitroparaffins; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase