Aryl-alcohol Oxidase Involved in Lignin Degradation: A MECHANISTIC STUDY BASED ON STEADY AND PRE-STEADY STATE KINETICS AND PRIMARY AND SOLVENT ISOTOPE EFFECTS WITH TWO ALCOHOL SUBSTRATES*

AUTOR(ES)

Ferreira, Patricia

FONTE

American Society for Biochemistry and Molecular Biology

RESUMO

Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O2 reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and pre-steady state conditions (the latter showing ∼9-fold slower enzyme reduction when α-bideuterated substrates were used, and ∼13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate α-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.

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