Substrate specificity of a nitroalkane-oxidizing enzyme

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of hydrogen peroxide and nitrite. The substrate specificity of the FAD-containing enzyme has been determined as a probe of the active site structure. Nitroalkane oxidase is active on primary and secondary nitroalkanes, with a marked preference for unbranched primary nitroalkanes. The V/K values for primary nitroalkanes increase with increasing length of the alkyl chain, reaching a maximum with 1-nitrobutane, suggesting a hydrophobic binding site sufficient to accommodate a four carbon chain. Each methylene group of the substrate contributes ~2.6 kcal mol^-1 in binding energy. The V/K values for substrates containing a hydroxyl group are two orders of magnitude smaller than those of the corresponding nitroalkanes, also consistent with a hydrophobic binding site. 3-Nitro-1-propionate is a competitive inhibitor with a K(is) value of 3.1 ± 0.2 mM.