René Ullrich

New and classic families of secreted fungal heme peroxidases.

Heme-containing peroxidases secreted by fungi are a fascinating group of biocatalysts with various ecological and biotechnological implications. For example, they are involved in the biodegradation of lignocelluloses and lignins and participate in the bioconversion of other diverse recalcitrant compounds as well as in the natural turnover of humic substances and organohalogens. The current review focuses on the most recently discovered and novel types of heme-dependent peroxidases, aromatic peroxygenases (APOs), and dye-decolorizing peroxidases (DyPs), which catalyze remarkable reactions such as peroxide-driven oxygen transfer and cleavage of anthraquinone derivatives, respectively, and represent own separate peroxidase superfamilies. Furthermore, several aspects of the “classic” fungal heme-containing peroxidases, i.e., lignin, manganese, and versatile peroxidases (LiP, MnP, and VP), phenol-oxidizing peroxidases as well as chloroperoxidase (CPO), are discussed against the background of recent scientific developments.

Enzymatic hydroxylation of aromatic compounds

Selective hydroxylation of aromatic compounds is among the most challenging chemical reactions in synthetic chemistry and has gained steadily increasing attention during recent years, particularly because of the use of hydroxylated aromatics as precursors for pharmaceuticals. Biocatalytic oxygen transfer by isolated enzymes or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. This review gives an overview of the different enzymes and mechanisms used to introduce oxygen atoms into aromatic molecules using either dioxygen (O2) or hydrogen peroxide (H2O2) as oxygen donors or indirect pathways via free radical intermediates. In this context, the article deals with Rieske-type and α-keto acid-dependent dioxygenases, as well as different non-heme monooxygenases (di-iron, pterin, and flavin enzymes), tyrosinase, laccase, and hydroxyl radical generating systems. The main emphasis is on the heme-containing enzymes, cytochrome P450 monooxygenases and peroxidases, including novel extracellular heme-thiolate haloperoxidases (peroxygenases), which are functional hybrids of both types of heme-biocatalysts.

Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes

ABSTRACT Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H2O2 (2 mM) for optimum activity. The apparent Km values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H2O2 were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.

Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type—the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago—has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV–Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as “self-sufficient” peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.

The haloperoxidase of the agaric fungus Agrocybe aegerita hydroxylates toluene and naphthalene

The mushroom Agrocybe aegerita secretes a peroxidase (AaP) that catalyzes halogenations and hydroxylations. Phenol was brominated to 2‐ and 4‐bromophenol (ratio 1:4) and chlorinated to a lesser extent to 2‐chlorophenol. The purified enzyme was found to oxidize toluene via benzyl alcohol and benzaldehyde into benzoic acid. A second fraction of toluene was hydroxylated to give p‐cresol as well as o‐cresol and methyl‐p‐benzoquinone. The UV–Vis absorption spectrum of purified AaP showed high similarity to a resting state cytochrome P450 with the Soret band at 420 nm and additional maxima at 278, 358, 541 and 571 nm; the AaP CO‐complex had a distinct absorption maximum at 445 nm that is characteristic for heme‐thiolate proteins. AaP regioselectively hydroxylated naphthalene to 1‐naphthol and traces of 2‐naphthol (ratio 36:1). H2O2 was necessarily required for AaP function and hence the hydroxylations catalyzed by AaP can be designated as peroxygenation and the enzyme as an extracellular peroxygenase.

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions—for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine—which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes’ affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO’s widespread occurrence in the fungal kingdom.

Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l−1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l−1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the “blue copper oxidases”. The enzyme’s pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (Km, kcat): 63 μM, 21 s−1 for ABTS, 4 μM, 5 s−1 for syringaldazine, 1,026 μM, 15 s−1 for DMP and 4307 μM, 159 s−1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.

The Coprophilous Mushroom Coprinus radians Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation

ABSTRACT Coprophilous and litter-decomposing species (26 strains) of the genus Coprinus were screened for peroxidase activities by using selective agar plate tests and complex media based on soybean meal. Two species, Coprinus radians and C. verticillatus, were found to produce peroxidases, which oxidized aryl alcohols to the corresponding aldehydes at pH 7 (a reaction that is typical for heme-thiolate haloperoxidases). The peroxidase of Coprinus radians was purified to homogeneity and characterized. Three fractions of the enzyme, CrP I, CrP II, and CrP III, with molecular masses of 43 to 45 kDa as well as isoelectric points between 3.8 and 4.2, were identified after purification by anion-exchange and size exclusion chromatography. The optimum pH of the major fraction (CrP II) for the oxidation of aryl alcohols was around 7, and an H2O2 concentration of 0.7 mM was most suitable regarding enzyme activity and stability. The apparent Km values for ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)], 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, and H2O2 were 49, 342, 635, 88, and 1,201 μM, respectively. The N terminus of CrP II showed 29% and 19% sequence identity to Agrocybe aegerita peroxidase (AaP) and chloroperoxidase, respectively. The UV-visible spectrum of CrP II was highly similar to that of resting-state cytochrome P450 enzymes, with the Soret band at 422 nm and additional maxima at 359, 542, and 571 nm. The reduced carbon monoxide complex showed an absorption maximum at 446 nm, which is characteristic of heme-thiolate proteins. CrP brominated phenol to 2- and 4-bromophenols and selectively hydroxylated naphthalene to 1-naphthol. Hence, after AaP, CrP is the second extracellular haloperoxidase-peroxygenase described so far. The ability to extracellularly hydroxylate aromatic compounds seems to be the key catalytic property of CrP and may be of general significance for the biotransformation of poorly available aromatic substances, such as lignin, humus, and organopollutants in soil litter and dung environments. Furthermore, aromatic peroxygenation is a promising target of biotechnological studies.

Oxidations catalyzed by fungal peroxygenases.

The enzymatic oxyfunctionalization of organic molecules under physiological conditions has attracted keen interest from the chemical community. Unspecific peroxygenases (EC 1.11.2.1) secreted by fungi represent an intriguing enzyme type that selectively transfers peroxide-borne oxygen with high efficiency to diverse substrates including unactivated hydrocarbons. They are glycosylated heme-thiolate enzymes that form a separate superfamily of heme proteins. Among the catalyzed reactions are hydroxylations, epoxidations, dealkylations, oxidations of organic hetero atoms and inorganic halides as well as one-electron oxidations. The substrate spectrum of fungal peroxygenases and the product patterns show similarities both to cytochrome P450 monooxygenases and classic heme peroxidases. Given that selective oxyfunctionalizations are among the most difficult to realize chemical reactions and that respectively transformed molecules are of general importance in organic and pharmaceutical syntheses, it will be worth developing peroxygenase biocatalysts for industrial applications.

Selective hydroxylation of alkanes by an extracellular fungal peroxygenase

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.