Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita

Abstract

Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p-benzoquinone, catechol and o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone, respectively. Using H218O2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p-benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (kcat, Km) for the peroxygenation of benzene were estimated to be around 8 s-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.

Photoenzymatic Hydroxylation of Ethylbenzene Catalyzed by Unspecific Peroxygenase: Origin of Enzyme Inactivation and the Impact of Light Intensity and Temperature

“…[2,3] For the selective oxyfunctionalization of CÀ HÀ and C=Cbonds, Unspecific Peroxygenases (UPOs, E.C. 1.11.2.1) are promising enzymes catalyzing a variety of oxidation reactions [4][5][6][7][8][9][10] like selective hydroxylations, [11][12][13][14] which can be used for example for the synthesis of human drug metabolites. [15][16][17] Particularly, the UPO from Agrocybe aegerita (AaeUPO) [9] available from recombinant fermentation in Pichia pastoris [18,19] is an attractive catalyst.…”

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

“…Despite these advantages, the lack of suitable directedevolution platforms with which to enhance UPO's catalytic properties has limited the exploitation of this versatile biocatalyst. The directed-evolution process presented here describes for the first time an attractive pathway through which ad hoc UPO variants can be tailored for use in several industrial reactions, such as alkane hydroxylation and the transformation of benzene into phenol and naphthalene into naphthol (51,52).…”

Search, engineering, and applications of new oxidative biocatalysts

“…Benzene hydroxylation by UPO to obtain phenol, and subsequent phenol hydroxylation to obtain p ‐hydroquinone (and catechol) were evaluated for their industrial feasibility, given the large markets of both compounds in plastic manufacture, photographic chemicals, and other products. The monohydroxylation and dihydroxylation reactions by the peroxygenase activity of UPO were analyzed in detail, starting with the epoxide that rearomatizes yielding phenol, together with the competing one‐electron oxidation (due to the additional peroxidase activity of UPO) yielding phenoxyl radicals, which can disproportionate to undesirable quinones and form coupling products (Fig. ).…”

“…Scheme for the epoxidation/hydroxylation and further oxidation of benzene (1) and its following products phenol (3) and p ‐hydroquinone (4) by UPO via the benzene epoxide intermediate (2) (in the same way, catechol may be oxidized to o ‐benzoquinone). Adapted from Karich et al …”