Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase

Fungal AAO (aryl-alcohol oxidase) provides H₂O₂ for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, during oxidation of some alcohols, AAO forms more than a stoichiometric number of H₂O₂ molecules with respect to the amount of aldehyde detected due to a double reaction that involves aryl-aldehyde oxidase activity. The latter reaction was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady- and presteady state kinetic constants, together with the chromatographic results, revealed that the presence of substrate electron-withdrawing or electron-donating substituents had a strong influence on activity; the highest activity was with p-nitrobenzaldehyde and halogenated aldehydes and the lowest with methoxylated aldehydes. Moreover, activity was correlated to the aldehyde hydration rates estimated by ¹H-NMR. These findings, together with the absence in the AAO active site of a residue able to drive oxidation via an aldehyde thiohemiacetal, suggested that oxidation mainly proceeds via the gem-diol species. The reaction mechanism (with a solvent isotope effect, ^2H₂Ok_red, of approx. 1.5) would be analogous to that described for alcohols, the reductive half-reaction involving concerted hydride transfer from the α-carbon and proton abstraction from one of the gem-diol hydroxy groups by a base. The existence of two steps of opposite polar requirements (hydration and hydride transfer) explains some aspects of aldehyde oxidation by AAO. Site-directed mutagenesis identified two histidine residues strongly involved in gem-diol oxidation and, unexpectedly, suggested that an active-site tyrosine residue could facilitate the oxidation of some aldehydes that show no detectable hydration. Double alcohol and aldehyde oxidase activities of AAO would contribute to H₂O₂ supply by the enzyme.