Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase

Abstract

Many litter-decay fungi secrete heme-thiolate peroxygenases that oxidize various organic chemicals, but little is known about the role or mechanism of these enzymes. We found that the extracellular peroxygenase of Agrocybe aegerita catalyzed the H₂O₂-dependent cleavage of environmentally significant ethers, including methyl t-butyl ether, tetrahydrofuran, and 1,4-dioxane. Experiments with tetrahydrofuran showed the reaction was a two-electron oxidation that generated one aldehyde group and one alcohol group, yielding the ring-opened product 4-hydroxybutanal. Investigations with several model substrates provided information about the route for ether cleavage: (a) steady-state kinetics results with methyl 3,4-dimethoxybenzyl ether, which was oxidized to 3,4-dimethoxybenzaldehyde, gave parallel double reciprocal plots suggestive of a ping-pong mechanism (K_m(peroxide), 1.99 ± 0.25 mM; K_m(ether), 1.43 ± 0.23 mM; k_cat, 720 ± 87 s^-1), (b) the cleavage of methyl 4-nitrobenzyl ether in the presence of H₂¹⁸O₂ resulted in incorporation of ¹⁸O into the carbonyl group of the resulting 4-nitrobenzaldehyde, and (c) the demethylation of 1-methoxy-4-trideuteromethoxybenzene showed an observed intramolecular deuterium isotope effect [(k_Hk_D)_obs] of 11.9 ± 0.4. These results suggest a hydrogen abstraction and oxygen rebound mechanism that oxidizes ethers to hemiacetals, which subsequently hydrolyze. The peroxygenase appeared to lack activity on macromolecular ethers, but otherwise exhibited a broad substrate range. It may accordingly have a role in the biodegradation of natural and anthropogenic low molecular weight ethers in soils and plant litter.

Citation

Kinne, Matthias; Poraj-Kobielska, Marzena; Ralph, Sally A.; Ullrich, Rene; Hofrichter, Martin; Hammel, Kenneth E. 2009. Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase. Journal of Biological Chemistry. 284(43): 29343-29349.