Abstract
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 O(2) 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 approximately 9-fold slower enzyme reduction when alpha-bideuterated substrates were used, and approximately 13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate alpha-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.
Highlights
Wood and other lignocellulosic materials are the main source of renewable materials in earth
The Aryl-alcohol oxidase (AAO) steady state kinetic parameters, obtained by varying both O2 and alcohol substrate concentrations, are consistent with a ternary complex mechanism (Scheme 1) with O2 reacting with the reduced enzyme before release of the aldehyde product
AAO appears to follow a similar sequential mechanism with regard to choline oxidase, a glucose-methanolcholine oxidases (GMC) oxidoreductase oxidizing alcohol substrates to the corresponding aldehydes and acids [17, 21], while a ping-pong mechanism appears to apply for glucose oxidase [19]
Summary
Chemicals—p-Anisyl alcohol, 2,4-hexadien-1-ol, p-anisic acid, 3-fluorobenzyl alcohol, sodium deuteroxide, and deuterium oxide (99.9%) were purchased from Sigma-Aldrich. p-[␣2H2]anisyl alcohol ([1,1-2H2]1-(4Ј-methoxyphenyl)-methanol) and [␣-2H2]2,4-hexadien-1-ol ([1,1-2H2]2,4-hexadien-1-ol) were synthesized at the Instituto de Ciencia de Materiales de Aragon (CSIC-UZ, Zaragoza, Spain). Chemicals—p-Anisyl alcohol, 2,4-hexadien-1-ol, p-anisic acid, 3-fluorobenzyl alcohol, sodium deuteroxide, and deuterium oxide (99.9%) were purchased from Sigma-Aldrich. Steady state kinetic measurements were monitored spectrophotometrically by oxidation of the alcohols (p-anisyl alcohol or 2,4-hexadien-1-ol) to the corresponding aldehydes [17]. Two-substrate steady state kinetic measurements were performed varying either the concentrations of the alcohol or O2 as described under supplemental materials (see supplemental Eqs. S1 and S2). The pH (4 –9) dependence of AAO activity was studied at 25 °C with p-anisyl alcohol as substrate under different O2 concentrations. The inhibition of AAO activity by p-anisic acid was studied at 25 °C, under atmospheric O2 concentration, using veratryl alcohol as substrate. Buffers used were the above indicated for steady state kinetics. Kinetic parameters (of steady and pre-steady states) for AAO oxidation of p-anisyl alcohol and 2,4-hexadien-1-ol. Assays were performed in 100 mM phosphate, pH 6, at 12 °C
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