Giovanni Gadda

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 λmax 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 (∼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.

Identification of a cysteine residue in the active site of nitroalkane oxidase by modification with N-ethylmaleimide.

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of primary or secondary nitroalkanes to the corresponding aldehydes or ketones with production of hydrogen peroxide and nitrite. The enzyme is irreversibly inactivated by treatment with N-ethylmaleimide at pH 7. The inactivation is time-dependent and shows first-order kinetics for three half-lives. The second-order rate constant for inactivation is 3.4 ± 0.06 m − 1min− 1. The competitive inhibitor valerate protects the enzyme from inactivation, indicating an active site-directed modification. Comparison of tryptic maps of enzyme treated withN-[ethyl-1-14C]maleimide in the absence and presence of valerate shows a single radioactive peptide differentially labeled in the unprotected enzyme. The sequence of this peptide was determined to be LLNEVMCYPLFDGGNIGLR using Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The cysteine residue was identified as the site of alkylation by ion trap mass spectrometry.

Evidence for an Essential Arginine in the Flavoprotein Nitroalkane Oxidase

The flavoprotein nitroalkane oxidase from the fungus Fusarium oxysporum catalyzes the oxidative denitrification of primary or secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. The enzyme is inactivated in a time-dependent fashion upon treatment with the arginine-directed reagents phenylglyoxal, 2,3-butanedione, and cyclohexanedione. The inactivation shows first order kinetics with all reagents. Valerate, a competitive inhibitor of the enzyme, fully protects the enzyme from inactivation, indicating that modification is active site directed. The most rapid inactivation is seen with phenylglyoxal, with a kinact of 14.3 ± 1.1 M−1 min−1 in phosphate buffer at pH 7.3 and 30 °C. The lack of increase in the enzymatic activity of the phenylglyoxal-inactivated enzyme after removing the unreacted reagent by gel filtration is consistent with inactivation being due to co-valent modification of the enzyme. A possible role for an active site arginine in substrate binding is discussed.