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 H(2)O(2) 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 H(2)O(2)-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 H(2)(18)(2) resulted in complete incorporation of (18)O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D(7) 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 × 10(11) 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 (> C(16)) 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.

Fig. 1

General hydroxylation reactions catalyzed by the peroxygenase from A. aegerita.

Fig. 2

GC/MS analysis of enantiomers of 2-alcohols and 3-alcohols produced during peroxygenase-catalyzed hydroxylation of (A) n-hexane (H) and (B) n-octane (O). The alcohols were measured as their trifluoroacetic acid esters.

Fig. 3

Incorporation of ¹⁸O from H₂¹⁸O₂ into the alcohol group of cyclohexanol after hydroxylation of cyclohexane by A. aegerita peroxygenase. Upper: mass spectrum of the product obtained with natural abundance H₂O₂. Structural assignments for m/z values are as follows: [M]⁺, 100; [M-C₃H₇]⁺, 57; [M-C₂H₅-C₂H₄]⁺, 44 [37]. Lower: mass spectrum of the product obtained with 90 atom% H₂¹⁸O₂.

Fig. 4

Preferential hydroxylation by A. aegerita peroxygenase of the nondeuterated ω-1 and ω-2 carbons in n-hexane-1,1,1,2,2,3,3-D₇. Upper: mass spectrum of the 2-hexanol-D₇ and 2-hexanol-D₆ trimethylsilylether derivatives obtained from the oxidation of natural abundance n-hexane-1,1,1,2,2,3,3-D₇. Lower: mass spectrum of the 3-hexanol-D₇ and 3-hexanol-D₆ trimethylsilylether derivatives obtained from the oxidation of natural abundance n-hexane-1,1,1,2,2,3,3-D₇. Each mass spectrum shown is one of three used to calculate the observed mean intramolecular isotope effect.

Fig. 5

GC/MS total ion current chromatogram in the region of the alcohols produced from the hydroxylation of norcarane by A. aegerita peroxygenase. In addition, traces of desaturation products and an epoxide were detected (data not shown).

Fig. 6

Hypothetical reaction cycle for alkane hydroxylation by A. aegerita peroxygenase, with cyclohexane shown as the oxidized substrate. Modified according to [11]. Note that the precise structure of the oxidized heme (compound I) of peroxygenase is still unclear and subject to current investigation. Furthermore, it is not yet known whether H₂O₂ or the alkane binds first to the enzyme.