Selective hydroxylation of alkanes by an extracellular fungal peroxygenase

Abstract

Fungal peroxygenases are novel extracellular heme‐thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H₂O₂ as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H₂O₂‐dependent hydroxylation of linear alkanes at the 2‐position and 3‐position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n‐heptane and n‐octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)‐enantiomer of the corresponding 3‐alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H₂¹⁸O₂ resulted in complete incorporation of ¹⁸O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n‐hexane‐1,1,1,2,2,3,3‐D₇ showed a large intramolecular deuterium isotope effect [(k_H/k_D)_obs] of 16.0 ± 1.0 for 2‐hexanol and 8.9 ± 0.9 for 3‐hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 10¹¹ s⁻¹. These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long‐chain alkanes (> C₁₆) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane‐containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood.