Kinetic and chemical characterization of aldehyde oxidation by fungal aryl-alcohol oxidase
Résumé
Fungal aryl-alcohol oxidase (AAO) provides H2O2 for lignin biodegradation. AAO is active on benzyl alcohols that are oxidized to aldehydes. However, the H2O2 formed from some of them was more than stoichiometric with respect to the aldehyde detected. This was due to a double reaction that involves aryl-aldehyde oxidase activity. The latter was investigated using different benzylic aldehydes, whose oxidation to acids was demonstrated by GC-MS. The steady and pre-steady state kinetic constants together with the chromatographic results revealed a strong influence of substrate electron withdrawing/donating substituents on activity, being the highest on p-nitrobenzaldehyde and halogenated aldehydes and the lowest on 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 solvent isotope effect of D2Okred ~1.5) would be analogous to that described for alcohols, the reductive half-reaction involving concerted hydride transfer from α-carbon, and proton abstraction from one of the gem-diol hydroxyls 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 histidines strongly involved in gem-diol oxidation and, unexpectedly, suggested that an active-site tyrosine could facilitate the oxidation of some aldehydes showing no detectable hydration. Double alcohol and aldehyde oxidase activities of AAO would contribute to H2O2 supply by the enzyme.
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