Characterization of the active fad-containing form of nitroalkane oxidase

Giovanni Gadda', Paul F Fitzpatrick

Research output: Contribution to journalArticle

Abstract

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes or ketones, producing H2Q2 and nitrite. The neutral form of the nitroalkane is required for catalysis, making this enzyme unique among flavoenzyme oxidases. NAO is purified with the flavin in the form of an inactive 5-nitrobutyl-FAD; this has been converted to the active FAD-containing form for characterization. AI pH 8.0 and 30 °C with nitroethane as substrate, the Vmax value is 700 min-l and the Km value 2.9 mM. Benzoate binds to NAO as shown by changes in the absorbance spectrum of the enzyme with a Kd value of 1.0 mM; kinetically, benzoate is a competitive inhibitor versus nitroethane. The enzyme reacts with sulfite with a Kj value of 13.8 mM. Despite these similarities to D-amino acid oxidase, NAO is not active with alanine or glycine as substrates. The enzyme has a low pKa value of 8.4 for the N(3) position of FAD. The apoprotein is not active; half-maximal activity occurs with 14.4 (iM FAD. No semiquinone has been observed upon anaerobic reduction.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997
Externally publishedYes

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Flavin-Adenine Dinucleotide
Benzoates
benzoates
Enzymes
enzymes
D-Amino-Acid Oxidase
Sulfites
apoproteins
flavins
Apoproteins
sulfites
Fusarium
Substrates
Nitrites
Fusarium oxysporum
ketones
Ketones
Catalysis
catalytic activity
glycine (amino acid)

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Characterization of the active fad-containing form of nitroalkane oxidase. / Gadda', Giovanni; Fitzpatrick, Paul F.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

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AB - Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes or ketones, producing H2Q2 and nitrite. The neutral form of the nitroalkane is required for catalysis, making this enzyme unique among flavoenzyme oxidases. NAO is purified with the flavin in the form of an inactive 5-nitrobutyl-FAD; this has been converted to the active FAD-containing form for characterization. AI pH 8.0 and 30 °C with nitroethane as substrate, the Vmax value is 700 min-l and the Km value 2.9 mM. Benzoate binds to NAO as shown by changes in the absorbance spectrum of the enzyme with a Kd value of 1.0 mM; kinetically, benzoate is a competitive inhibitor versus nitroethane. The enzyme reacts with sulfite with a Kj value of 13.8 mM. Despite these similarities to D-amino acid oxidase, NAO is not active with alanine or glycine as substrates. The enzyme has a low pKa value of 8.4 for the N(3) position of FAD. The apoprotein is not active; half-maximal activity occurs with 14.4 (iM FAD. No semiquinone has been observed upon anaerobic reduction.

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