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 be en determined as a probe of the active site structure. Nitroalkane oxidase is active on primary and secondary nitroalkanes, with a marked preference f or unbranched primary nitroalkanes. The V/K values for primary nitroalkanes increase with increasing length of the alkyl chain, reaching a maximum wit h 1-nitrobutane, suggesting a hydrophobic binding site sufficient to accomm odate a four carbon chain. Each methylene group of the substrate contribute s similar to 2.6 kcal mol(-1) in binding energy. The V/K values for substra tes containing a hydroxyl group are two orders of magnitude smaller than th ose of the corresponding nitroalkanes, also consistent with a hydrophobic b inding site. 3-Nitro-1-propionate is a competitive inhibitor with a K-is va lue of 3.1 +/- 0.2 mM. (C) 1999 Academic Press.