TY - GEN A1 - Kinne, Matthias A1 - Poraj-Kobielska, Marzena A1 - Ullrich, René A1 - Nousiainen, Paula A1 - Sipilä, Jussi A1 - Scheibner, Katrin A1 - Hammel, Kenneth E. A1 - Hofrichter, Martin T1 - Oxidative cleavage of non-phenolic b-O-4 lignin model dimers by an extracellular aromatic peroxygenase T2 - Holzforschung N2 - The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita catalyzed the H2O2-dependent cleavage of non-phenolic arylglycerol-b-aryl ethers (b-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)pro- pane-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 H218O2 and H218O at different positions into the products 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 peroxyge- nases may be involved in the bioconversion of methoxylated plant ingredients originating from lignin or other sources. KW - Agrocybe aegerita KW - hydroxylation KW - lignin model compound KW - 0-dealkylation KW - peroxidase KW - peroxygenase Y1 - 2011 U6 - https://doi.org/10.1515/HF.2011.057 SN - 1437-434X VL - 65 IS - 5 SP - 673 EP - 679 ER - TY - GEN A1 - Yarman, Aysu A1 - Peng, Lei A1 - Wu, Yunhua A1 - Bandodkar, Amay A1 - Gajovic-Eichelmann, Nenad A1 - Wollenberger, Ulla A1 - Hofrichter, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Scheller, Frieder W. T1 - Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors T2 - Bioanalytical Reviews N2 - Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes. KW - Cytochrome P450 KW - Aromatic peroxygenase KW - Microperoxidase KW - Biosensors Y1 - 2011 UR - http://link.springer.com/article/10.1007%2Fs12566-011-0023-4 U6 - https://doi.org/10.1007/s12566-011-0023-4 SN - 1867-2094 VL - 3 IS - 2-4 SP - 67 EP - 94 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Kinne, Matthias A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Kayser, Gernot A1 - Hammel, Kenneth E. A1 - Hofrichter, Martin T1 - Preparation of human drug metabolites using fungal peroxygenases T2 - Biochemical Pharmacology N2 - 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. KW - Peroxidase KW - Peroxygenation KW - Hydroxylation KW - O-Dealkylation KW - N-Dealkylation KW - Cytochrome P450 Y1 - 2011 UR - http://www.sciencedirect.com/science/article/pii/S0006295211004035 U6 - https://doi.org/10.1016/j.bcp.2011.06.020 SN - 1873-2968 VL - 82 IS - 7 SP - 789 EP - 796 ER - TY - GEN A1 - Peng, Lei A1 - Wollenberger, Ulla A1 - Kinne, Matthias A1 - Hofrichter, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Fischer, Anna A1 - Scheller, Frieder W. T1 - Peroxygenase based sensor for aromatic compounds T2 - Biosensors and Bioelectronics N2 - 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. KW - Peroxygenase KW - Direct electron transfer KW - Nanoparticles KW - Naphthalene biosensor KW - Bioelectrocatalysis Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?http://www.sciencedirect.com/science/article/pii/S0956566310004501 SN - 1873-4235 VL - 26 IS - 4 SP - 1432 EP - 1436 ER - TY - GEN A1 - Peng, Lei A1 - Wollenberger, Ulla A1 - Hofrichter, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Scheller, Frieder W. T1 - Bioelectrocatalytic properties of Agrocybe aegerita peroxygenase T2 - Electrochimica Acta N2 - 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. KW - Peroxygenase KW - Cytochrome P450s KW - Horseradish peroxidase KW - 4-Nitrophenol KW - Agrocybe aegerita Y1 - 2010 UR - http://www.sciencedirect.com/science/article/pii/S0013468610000149 U6 - https://doi.org/10.1016/j.electacta.2009.12.065 SN - 0013-4686 VL - 55 IS - 27 SP - 7809 EP - 7813 ER - TY - GEN A1 - Pecyna, Marek J. A1 - Ullrich, René A1 - Bittner, Britta A1 - Clemens, André A1 - Scheibner, Katrin A1 - Schubert, Roland A1 - Hofrichter, Martin T1 - Molecular characterization of aromatic peroxygenase from Agrocybe aegerita T2 - Applied Microbiology and Biotechnology N2 - 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. KW - Peroxygenase KW - Chloroperoxidase KW - Cytochrome P450 KW - Heme-thiolate KW - Oxygenation KW - Coprinellus Y1 - 2009 UR - http://link.springer.com/article/10.1007%2Fs00253-009-2000-1 U6 - https://doi.org/10.1007/s00253-009-2000-1 SN - 1432-0614 VL - 84 IS - 5 SP - 885 EP - 897 ER - TY - GEN A1 - Kinne, Matthias A1 - Poraj-Kobielska, Marzena A1 - Aranda, Elisabet A1 - Ullrich, René A1 - Hammel, Kenneth E. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase T2 - Bioorganic & Medicinal Chemistry Letters N2 - 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. KW - Peroxidase KW - Peroxygenase KW - Oxygenase KW - Cytochrome P450 KW - Hydroxylation KW - 5-Hydroxypropranolol KW - Propranolol KW - 4′-Hydroxydiclofenac KW - Diclofenac KW - Ascorbic acid Y1 - 2009 UR - http://www.sciencedirect.com/science/article/pii/S0960894X09005071 U6 - https://doi.org/10.1016/j.bmcl.2009.04.015 SN - 1464-3405 VL - 19 IS - 11 SP - 3085 EP - 3087 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Dolge, Christoph A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation T2 - Applied Microbiology and Biotechnology N2 - 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. KW - Peroxidase KW - Oxygenation KW - Hydroxylation KW - P450 KW - Naphthol Y1 - 2009 UR - http://link.springer.com/article/10.1007%2Fs00253-008-1704-y U6 - https://doi.org/10.1007/s00253-008-1704-y SN - 0175-7598 VL - 81 IS - 6 SP - 1071 EP - 1076 ER - TY - GEN A1 - Kinne, Matthias A1 - Ullrich, René A1 - Hammel, Kenneth E. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Regioselective preparation of (R)-2-(4-Hydroxyphenoxy)propionic acid with a fungal peroxygenase T2 - Tetrahedron Letters N2 - 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. KW - Peroxidase KW - Peroxygenase KW - Oxygenase KW - Cytochrome P450 KW - Hydroxylation KW - 2-(4-Hydroxyphenoxy)propionic acid KW - Ascorbic acid Y1 - 2008 UR - http://www.sciencedirect.com/science/article/pii/S0040403908014421 U6 - https://doi.org/10.1016/j.tetlet.2008.07.152 SN - 1873-3581 VL - 49 IS - 41 SP - 5950 EP - 5953 ER - TY - GEN A1 - Gröbe, Glenn A1 - Ullrich, René A1 - Pecyna, Marek J. A1 - Kapturska, Danuta A1 - Friedrich, Stephanie A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula T2 - AMB Express N2 - An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities. KW - Peroxygenase KW - Peroxidase KW - Basidiomycota KW - Cytochrome P450 KW - Bioreactor Y1 - 2011 UR - http://www.amb-express.com/content/1/1/31 SN - 2191-0855 ER -