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.
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 nm 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 lambdamax 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 pKa 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 ( approximately 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 pKa 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.
Gadda
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Department of Biochemistry and Biophysics, Texas A & M University, College Station, Texas 77843-2128, USA.
Edmondson
R D
RD
Russell
D H
DH
Fitzpatrick
P F
PF
D002942:Circular Dichroism; D049308:Dioxygenases; D004980:Ethane / Q000031:analogs & derivatives / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009595:Nitroparaffins / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry*; D011487:Protein Conformation; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet
D005415:Flavins; D009595:Nitroparaffins; D005182:Flavin-Adenine Dinucleotide; C014705:nitroethane; D010105:Oxygenases; D049308:Dioxygenases; C021876:2-nitropropane dioxygenase; D004980:Ethane