Prof. Enzymtechnologie
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- Peroxygenase (10)
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Institute
- FG Enzymtechnologie (21)
Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally-functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural-functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by ‘non-rational’ directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short-chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy-fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl-benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries.
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.
Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ 303 = 2,010 M−1 cm−1), and the apparent Michaelis–Menten (K m) and catalytic (k cat) constants for the reaction were estimated to be 320 μM and 166 s−1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.
Regioselective preparation of (R)-2-(4-Hydroxyphenoxy)propionic acid with a fungal peroxygenase
(2008)
The extracellular heme-thiolate peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of 2-phenoxypropionic acid (POPA) to give the herbicide precursor 2-(4-hydroxyphenoxy)propionic acid (HPOPA). The reaction proceeded regioselectively with an isomeric purity near 98%, and yielded the desired R-isomer of HPOPA with an enantiomeric excess of 60%. 18O-labeling experiments showed that the phenolic hydroxyl in HPOPA originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for a variety of organic oxidations.
Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A max 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using 18O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.
An extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4′-hydroxydiclofenac (4′-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4′-OHD in yields up to 20% and 65%, respectively. 18O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4′-OHD originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.
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.
A biosensor for detecting the aromatic substance 4-nitrophenol based on Agrocybe aegerita peroxygenase (AaP) immobilized with chitosan-stabilized gold nanoparticles is presented here. This biosensor measures the enzymatic product of 4-nitrophenol peroxygenation, 4-nitrocatechol, which is electrochemically detected in the presence of hydrogen peroxide. Cyclic voltammetry and amperometry were used to characterize the proposed biosensor. The linear range of the AaP biosensor for the detection of 4-nitrophenol was between 10 and 30 μM with a detection limit of 0.2 μM (based on the S/N = 3). The catalytic property of AaP to oxidize 4-nitrophenol was compared with two other heme proteins, a camphor-hydroxylating cytochrome P450 monooxygenase (P450cam, CYP101) and horseradish peroxidase (HRP). The results revealed that only AaP is capable of catalyzing the hydroxylation of 4-nitrophenol into 4-nitrocatechol. Consequently, AaP could be a particularly potent biocatalyst that may fill the gap between cytochrome P450s and common heme peroxidases.
We report on the redox behaviour of the peroxygenase from Agrocybe aegerita (AaeAPO) which has been electrostatically immobilized in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode. AaeAPO contains a covalently bound heme-thiolate as the redox active group that exchanges directly electrons with the electrode via the gold nanoparticles. The formal potential E ° ′ of AaeAPO in the gold nanoparticles-chitosan film was estimated to be −(286 ± 9) mV at pH 7.0. The heterogeneous electron transfer rate constant (ks) increases from 3.7 in the scan rate range from 0.2 to 3.0 V s−1 and level off at 63.7 s−1.
Furthermore, the peroxide-dependent hydroxylation of aromatic compounds was applied to develop a sensor for naphthalene and nitrophenol. The amperometric measurements of naphthalene are based on the indication of H2O2 consumption. For the chitosan-embedded gold nanoparticle system, the linear range extends from 4 to 40 μM naphthalene with a detection limit of 4.0 μM (S/N = 3) and repeatability of 5.7% for 40 μM naphthalene.
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.