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 [(kH/kD)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.

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

“…Thus, C−H hydrogen abstraction by APO-I will proceed to a protonated APO-II in a proton-coupled electron transfer event in which one electron will be transferred to the porphyrin radical cation of APO-I (29). It is at this stage that the incipient substrate radical can either rebound to form product alcohols or rearrange in the enzyme active site as has been observed (26).…”

“…These proteins are unrelated to CYP enzymes, according to their amino acid sequences, and are only distantly related to CPO, with about 30% sequence similarity and a similar tertiary structure (25). APO proteins have shown high activity for the oxygenation of aliphatic and aromatic hydrocarbons and a variety of other organic substrates, in sharp contrast to CPO (26). These APO proteins are unusually stable, heavily glycosylated, extracellular proteins that apparently serve to mobilize food sources for the growing organisms through oxidative degradation and to detoxify harmful compounds in their microenvironment.…”

Structural Basis of Substrate Conversion in a New Aromatic Peroxygenase

“…Further organic substrates of AaeAPO, both aromatics as well as aliphatics, were identified in close succession (10,12). AaeAPO turned out to be an accomplished catalyst for chemo-and stereoselective reactions, for example for the introduction of oxygen into hydrocarbons (10,(12)(13)(14). Alkanes such as heptane are hydroxylated in the 2-and the 3-positions with enantiomeric excesses up to 99.9% (Fig.…”

“…1). The enantioselectivity of alkane hydroxylation achieved with AaeAPO surpasses that of P450 oxygenases by far (12). Its catalytic versatility has been demonstrated for various substrates, such as naphthalenes, toluene, ethylbenzene, alkanes, alkenes, and saturated as well as unsaturated fatty acids (10,14).…”

Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

“…However, unlike P450s, which are intracellular enzymes whose activation often requires an auxiliary enzyme and a source of reducing power, the A. aegerita enzyme is a secreted protein; therefore, it is far more stable and requires only H 2 O 2 for activation (8). The A. aegerita peroxygenase has been shown to catalyze numerous interesting oxygenation reactions on aromatic compounds (10), and recently, the action of this enzyme on aliphatic compounds was demonstrated, expanding its biotechnological interest (11,12). The enzyme, first described as a haloperoxidase and later mostly referred to as aromatic peroxygenase (APO), now is named "unspecific peroxygenase" (UPO; EC 1.11.2.1).…”