Identification of a Hypothetical Protein from Podospora anserina as a Nitroalkane Oxidase

Abstract

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated an NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k cat /K m -pH profile with nitroethane shows a pK a of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k cat /K m value for nitroethane over two orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64 and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 Å, confirming its identification as an NAO.The flavoenzyme nitroalkane oxidase (NAO) from the soil fungus Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with the release of nitrite and the consumption of molecular oxygen to yield hydrogen peroxide (Scheme 1) (1,2). NAO is unusual, since it catalyzes substrate oxidation by removing a substrate proton to form a carbanion intermediate (3) (Scheme 2), whereas flavoproteins that oxidize carbonnitrogen and carbon-oxygen bonds typically catalyze cleavage of the substrate carbonhydrogen bond by removal of a hydride rather than a proton (4-6). The NAO reaction is initiated by abstraction of the α-proton from the neutral form of the nitroalkane by Asp402, the active site base (7), creating a nucleophilic nitroalkane anion (Scheme 2). The anion then attacks the N5 position of FAD to form an adduct; this eliminates nitrite to generate a cationic, electrophilic flavin imine that can be attacked by hydroxide (8)(9)(10)(11) product forms reduced FAD (7). In the more typical oxidative half-reaction, the reduced FAD is oxidized by molecular oxygen to form hydrogen peroxide (2). Release of products from the oxidized enzyme limits turnover with primary nitroalkanes, the best substrates (12). With the slow substrate nitroethane (13), formation of the substrate anion is rate-limiting for the reductive half-reaction (12).The proton transfer reaction between nitroethane and Asp402 catalyzed by F. oxysporum NAO provides an excellent opportunity for a study of the basis for enzymatic catalysis because the enzymatic process can be modeled by the reaction of nitroethane and acetate in water (14). Nitroalkanes represent a prototypical system for the study ...

Flavoprotein oxidases: classification and applications

“…The carbanion obtained forms a covalent adduct with the N5 of the flavin, which generates a cationic flavin imine. Attack by water leads to the reduced flavin and the final product, the aldehyde or ketone (Tormos et al 2010). Reoxidation of the reduced flavin cofactor by molecular oxygen completes the catalytic cycle.…”

Solvent isotope and viscosity effects on the steady‐state kinetics of the flavoprotein nitroalkane oxidase

“…Nitroalkane oxidase activity was first identified in the soil fungus Fusarium oxysporum (1), but has since been identified in other fungi (2). Extensive mechanistic and structural studies of the enzyme from F. oxysporum have been carried out to date, including determination of structures of reaction intermediates (3) and mutagenesis of active site residues (3–6).…”

Nitroalkane oxidase: Structure and mechanism

“…To identify additional NAOs a PSI-Blast [44] search of sequence databases was carried out using the sequence of F. oxysporum NAO. Eliminating any sequences in which the residue aligning with Asp402 was a glutamate allowed identification of putative NAOs [45]. All of the candidates were fungal proteins.…”