Nitroalkane oxidase (NAO) catalyzes the oxidation of nitroalkanes to the corresponding aldehydes and ketones with consumption of oxygen and production of nitrite and hydrogen peroxide (Figure 1). The enzyme is induced in Fusarium oxysporum in the presence of nitroalkanes and the fungus is able to grow on the nitrite released from the enzymatic reaction as the sole source of nitrogen. Primary nitroalkanes are the best substrates for NAO, but it can oxidize a variety of nitroalkanes (1).

The catalytic mechanism of NAO can be divided into reductive and oxidative half-reactions (2). In the reductive half-reaction (Figure 2), a catalytic base in the active site removes a proton from the nitroalkane substrate to form a carbanion (3). This species then attacks the N5 position of the flavin to form an adduct that rearranges with release of nitrite. As part of the catalytic cycle, this intermediate reacts with hydroxide (pathway A) to ultimately form the aldehyde or ketone product and reduced flavin. However, it can also react with other nucleophiles, such as nitroalkane anions, to form relatively stable and inactive adducts (pathway B). Upon purification of the native enzyme from F. oxysporum, the flavin adenine dinucleotide (FAD) cofactor has been isolated and identified as the 5-nitrobutyl adduct shown in Figure 2, providing support for this mechanism (3). In the oxidative half-reaction, the reduced flavin reacts with oxygen to yield oxidized flavin and hydrogen peroxide.

Our current work is aimed at defining the mechanism of NAO and relating structure to function. By using alternate substrates, different isotopes, and various small molecules that both inhibit and activate NAO, we can resolve different steps in the catalytic mechanism in both steady-state and transient kinetic experiments (2, 4, 5). The gene for NAO has recently been cloned and the recombinant enzyme can be overexpressed in Escherichia coli (6). As a result, we have begun expressing and characterizing mutant enzymes in order to understand the catalytic role of individual amino acids. Previous chemical modification studies have suggested that certain residues are important (7, 8), but we are also collaborating with Akanksha Nagpal and Dr. Allen M. Orville at Georgia Tech in order to determine the three-dimensional structure of NAO.

Our greatest insights to date have derived from the homology between NAO and the family of flavoenzymes known as the acyl-CoA dehydrogenases (ACADs). A glutamate serves as the catalytic base in the medium and short chain ACADs and aligns with aspartate 402 in NAO (6). Although conservative mutation of this residue to glutamate causes a decrease in activity, mutation to either asparagine or alanine abolishes activity with the neutral substrates and is consistent with a role for aspartate 402 as the catalytic base (9, 10). Moreover, although NAO cannot oxidize acyl-CoA substrates and the ACADS cannot oxidize nitroalkanes, the high homology between these enzymes suggests that a minimal number of mutations would be required to generate the activity of the other enzymes. Thus, we have recently initiated directed evolution studies with the goal of generating an acyl-CoA oxidase specific for oleoyl-CoA or octanoyl-CoA from the gene for NAO. Conversely, we are also attempting to generate nitroalkane oxidase activity from the genes for the human medium chain ACAD and yafH, the only known ACAD in E. coli.

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