Reductive Half-Reaction of Nitroalkane Oxidase:  Effect of Mutation of the Active Site Aspartate to Glutamate^,

Abstract

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG ‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) 1 catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with consumption of oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). While several other flavoprotein oxidases are able to oxidize nitroalkanes, they require the preformed anion as a substrate in a clearly nonphysiological reaction (2). NAO is unique in that it uses the neutral forms of nitroalkanes as substrates (3); moreover, the enzyme is induced by growth of Fusarium oxysporum on nitroethane, consistent with nitroalkane oxidation being the physiological role of the enzyme (1). NAO can oxidize a broad range of primary and secondary nitroalkanes, although primary aliphatic nitroalkanes are the best substrates (4).As is the case with many flavoproteins, the reaction of NAO can be divided into reductive and oxidative half-reactions (5). In the reductive half-reaction, the nitroalkane substrate reacts with the oxidized flavin to generate the oxidized product and reduced flavin. The mechanism of this half-reaction (Scheme 2) has been proposed to involve removal of the substrate α-hydrogen as a proton, followed by attack of the resulting carbanion on the flavin (6). Loss of nitrite from the resulting adduct would generate an electrophilic species to which hydroxide adds (path a); this flavin adduct then decomposes to form reduced flavin and aldehyde or ketone. Evidence for this mechanism comes from the demonstration that an inactive 5-nitrobutyl-FAD adduct

Combined QM/MM and path integral simulations of kinetic isotope effects in the proton transfer reaction between nitroethane and acetate ion in water

“…50 This process is also catalyzed by nitroalkane oxidase in the initial step of the oxidation of nitroalkanes. 53 For the enzymatic process, the deprotonation step by Asp402 is rate-limiting with nitroethane as the substrate. Recently, Valley and Fitzpatrick determined the KIEs for the dideuterated substrate [1,1-2 H 2 ]nitroethane in nitroalkane oxidase, and the proton/deuteron abstraction reaction by an acetate ion in water in the absence of a catalyst.…”

“…It will be interesting to examine the effects of the enzyme active site on tunneling and the Swain-Schaad exponents. 53,54 …”

Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase,

“…Recombinant nitroalkane oxidase was expressed and purified as previously described (24). The D402E enzyme was purified as previously described (14). The codon for Arg409 was mutated to lysine, alanine, and glutamine using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), and the mutant enzymes were expressed and purified following the protocol for the wild-type enzyme (24).…”

“…As is generally the case with flavoproteins, the catalytic mechanism of NAO can be divided into oxidative and reductive half reactions (13,14). In the reductive half-reaction (Scheme 2), an active site base abstracts the α-proton from the neutral nitroalkane substrate to form a nitroalkane anion.…”

“…The role of Asp402 in NAO has been analyzed previously by site-directed mutagenesis. Mutagenesis to glutamate decreases the rate constant for abstraction of the nitroalkane proton by 15-fold (14), while mutagenesis to alanine or asparagine decreases it by at least 1000-fold (14).…”