Marzena Poraj-Kobielska

Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase

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 (Km(peroxide), 1.99 ± 0.25 mm; Km(ether), 1.43 ± 0.23 mm; kcat, 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 [(kH/kD)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.

Preparation of human drug metabolites using fungal peroxygenases.

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 3.1±0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.

A spectrophotometric assay for the detection of fungal peroxygenases.

Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε(425)=9,700 M(-1) cm(-1)), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε(514)=11,400 M(-1) cm(-1)). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.

Oxidative cleavage of non-phenolic ß-0-4 lignin model dimers by an extracellular aromatic peroxygenase

Abstract The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita catalyzed the H2O2-dependent cleavage of non-phenolic arylglycerol-β-aryl ethers (β-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)propane-1,3-diol, a recalcitrant dimeric lignin model compound that represents the major non-phenolic substructure in lignin, was selectively O-demethylated at the para-methoxy group to give formaldehyde and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol. The phenol moiety of the latter compound was then enzymatically oxidized into phenoxy radicals and a quinoid cation, which initiated the autocatalytic cleavage of the dimer and the formation of monomers such as 2-methoxy-1,4-benzoquinone and phenoxyl-substituted propionic acid. The introduction of 18O from H2 18O2 and H2 18O at different positions into the pro-ducts provided information about the routes of ether cleavage. Studies with a 14C-labeled lignin model dimer showed that more than 70% of the intermediates formed were further coupled to form polymers with molecular masses above 10 kDa. The results indicate that fungal aromatic peroxygenases may be involved in the bioconversion of methoxylated plant ingredients originating from lignin or other sources.

Side chain removal from corticosteroids by unspecific peroxygenase

Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichsteins substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzymes access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system.