Identification of the naturally occurring flavin of nitroalkane oxidase from Fusarium oxysporum as a 5-nitrobutyl-FAD and conversion of the enzyme to the active FAD-containing form

Giovanni Gadda, Ricky D. Edmondson, David H. Russell, Paul F. Fitzpatrick

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The UV-visible absorbance spectrum of the purified enzyme shows a single absorption peak at 336 nrn with an extinction coefficient of 7.4 mM-1 cm- 1. Upon denaturation of the enzyme at pH 7.0, a stoichiometric amount of FAD is released. The spectral properties of the enzyme as isolated are consistent with an N(5) adduct of the flavin. This is not due to a covalent linkage with the protein, since the free flavin adduct can be isolated from the enzyme at pH 2.1. The free flavin adduct shows an absorbance spectrum with a λ(max) at 346 nm (10.7 mM-1 cm-1) and is not fluorescent. Under alkaline conditions the free adduct decays, yielding FAD; the rate of this process is pH- dependent with a pK(α) of 7.4. Adduct decay is also observed with the native enzyme; in this case, however, the rate of decay is 160-fold slower (at pH 8.0) and not dependent on pH. During this process a large increase in enzymatic activity (~26-fold at pH 7.0) is observed, the rate of which is equal to the rate of flavin adduct conversion to FAD. Thus, the native flavin adduct is not active but can be converted to FAD, the active form of the flavin. Maximal activation is pH- and FAD-dependent; two groups with pK(α) values of 5.65 ± 0.25 and 8.75 ± 0.05 must be unprotonated and protonated, respectively. The m/z of the free flavin adduct is 103.0645 higher than that of FAD, as determined by matrix-assisted laser desorption ionization time- of-flight mass spectrometry. This corresponds to a molecule of nitrobutane linked to FAD. A mechanism is proposed for the formation in vivo of the nitrobutyl-FAD of nitroalkane oxidase.

Original languageEnglish (US)
Pages (from-to)5563-5570
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number9
DOIs
StatePublished - Feb 28 1997

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Identification of the naturally occurring flavin of nitroalkane oxidase from Fusarium oxysporum as a 5-nitrobutyl-FAD and conversion of the enzyme to the active FAD-containing form'. Together they form a unique fingerprint.

  • Cite this