TY - GEN A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Schramm, Marina A1 - Hofrichter, Martin A1 - König, Rosalie A1 - Quint, Stephan A1 - Kohlmann, Johannes A1 - Zuhse, Ralf A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases T2 - Journal of Fungi N2 - Antithrombotic thienopyridines, such as clopidogrel and prasugrel, are prodrugs that undergo a metabolic two-step bioactivation for their pharmacological efficacy. In the first step, a thiolactone is formed, which is then converted by cytochrome P450-dependent oxidation via sulfenic acids to the active thiol metabolites. These metabolites are the active compounds that inhibit the platelet P2Y12 receptor and thereby prevent atherothrombotic events. Thus far, described biocatalytic and chemical synthesis approaches to obtain active thienopyridine metabolites are rather complex and suffer from low yields. In the present study, several unspecific peroxygenases (UPOs, EC 1.11.2.1) known to efficiently mimic P450 reactions in vitro—but requiring only hydroperoxide as oxidant—were tested for biocatalytic one-pot syntheses. In the course of the reaction optimization, various parameters such as pH and reductant, as well as organic solvent and amount were varied. The best results for the conversion of 1 mM thienopyridine were achieved using 2 U mL−1 of a UPO from agaric fungus Marasmius rotula (MroUPO) in a phosphate-buffered system (pH 7) containing 5 mM ascorbate, 2 mM h−1 H2O2 and 20% acetone. The preparation of the active metabolite of clopidogrel was successful via a two-step oxidation with an overall yield of 25%. In the case of prasugrel, a cascade of porcine liver esterase (PLE) and MroUPO was applied, resulting in a yield of 44%. The two metabolites were isolated with high purity, and their structures were confirmed by MS and MS2 spectrometry as well as NMR spectroscopy. The findings broaden the scope of UPO applications again and demonstrate that they can be effectively used for the selective synthesis of metabolites and late-state diversification of organic molecules, circumventing complex multistage chemical syntheses and providing sufficient material for structural elucidation, reference material, or cellular assays. KW - Peroxygenase KW - clopidogrel Y1 - 2021 UR - https://www.mdpi.com/2309-608X/7/9/752 U6 - https://doi.org/10.3390/jof7090752 SN - 2309-608X VL - 7 IS - 9 SP - 1 EP - 17 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 - Friedrich, Stephanie A1 - Gröbe, Glenn A1 - Kluge, Martin A1 - Brinkmann, Tobias A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - Optimization of a biocatalytic process to gain (R)-1-phenylethanol by applying the software tool Sabento for ecological assessment during the early stages of development T2 - Journal of Molecular Catalysis : B, Enzymatic N2 - Ecological assessment using the software tool Sabento was conducted to compare different processes to gain the fine chemical (R)-1-phenylethanol from ethylbenzene. The software was applied during the biocatalytic process development using the unspecific peroxygenase (EC .11.2.1) of the fungus Agrocybe aegerita. The process could be systematically improved with respect to the ecological performance during process development. Compared to a modern chemical process and a further biotechnological process, it now reaches the best environmental key indicator. The software tool Sabento proved to be well suited to work out the most important factors determining the ecological burdens in the early stages of process development. KW - Ecological assessment KW - Biocatalytic process for (R)-1-phenylethanol KW - Unspecific peroxygenase (EC 1.11.2.1) KW - Agrocybe aegerita Y1 - 2014 UR - http://www.sciencedirect.com/science/article/pii/S1381117713002774 U6 - https://doi.org/10.1016/j.molcatb.2013.10.002 SN - 1381-1177 IS - 103 SP - 36 EP - 40 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Atzrodt, Jens A1 - Holla, Wolfgang A1 - Sandvoss, Martin A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases T2 - Journal of Labelled Compounds and Radiopharmaceuticals N2 - Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions. KW - Isotopic labeled synthesis KW - deuterium KW - 4'OH-diclofenac KW - paracetamol KW - phenacetin KW - lidocaine KW - unspecific/aromatic peroxygenase KW - EC 1.11.2.1; KW - human drug metabolites Y1 - 2013 UR - http://onlinelibrary.wiley.com/doi/10.1002/jlcr.3103/abstract;jsessionid=5453A37788468921BE8A03CABF2928DD.f03t02 U6 - https://doi.org/10.1002/jlcr.3103 SN - 1099-1344 VL - 56 IS - 9-10 SP - 513 EP - 519 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Formation of naphthalene hydrates in the enzymatic conversion of 1,2-dihydronaphthalene by two fungal peroxygenases and subsequent naphthalene formation T2 - Journal of Molecular Catalysis : B, Enzymatic N2 - The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2-dihydronaphthalene) displays a new activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal unspecific peroxygenases (UPOs) accounting for 16–19% of the overall turnover. These arene hydrates decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred during hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction. KW - Unspecific peroxygenase KW - Naphthalene hydrates KW - Aromatization KW - Oxygenation Y1 - 2014 UR - http://www.sciencedirect.com/science/article/pii/S1381117713002464 U6 - https://doi.org/10.1016/j.molcatb.2013.08.017 SN - 1381-1177 IS - 103 SP - 56 EP - 60 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita T2 - Green Chemistry N2 - Here we report on the stereoselective benzylic hydroxylation and C1-C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-[small beta]-methylstyrene oxide formation, an ee >99% and a TTN of 110 000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of 18O from H218O2 into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis. Y1 - 2012 UR - http://pubs.rsc.org/en/Content/ArticleLanding/2012/GC/C1GC16173C#!divAbstract SN - 1463-9270 IS - 2 SP - 440 EP - 446 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase T2 - Applied Microbiology and Biotechnology N2 - 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. KW - Hydroxylation KW - Peroxygenase Y1 - 2007 UR - http://link.springer.com/article/10.1007%2Fs00253-007-0942-8 U6 - https://doi.org/10.1007/s00253-007-0942-8 SN - 1432-0614 VL - 75 IS - 6 SP - 1473 EP - 1478 ER - TY - GEN A1 - Kiebist, Jan A1 - Holla, Wolfgang A1 - Heidrich, Johannes A1 - Poraj-Kobielska, Marzena A1 - Sandvoss, Martin A1 - Simonis, Reiner A1 - Gröbe, Glenn A1 - Atzrodt, Jens A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - One-pot synthesis of human metabolites of SAR548304 by fungal peroxygenases T2 - Bioorganic & Medicinal Chemistry N2 - Unspecific peroxygenases (UPOs, EC 1.11.2.1) have proved to be stable oxygen-transferring biocatalysts for H2O2-dependent transformation of pharmaceuticals. We have applied UPOs in a drug development program and consider the enzymatic approach in parallel to a conventional chemical synthesis of the human metabolites of the bile acid reabsorption inhibitor SAR548304. Chemical preparation of N,N-di-desmethyl metabolite was realized by a seven-step synthesis starting from a late precursor of SAR548304 and included among others palladium catalysis and laborious chromatographic purification with an overall yield of 27%. The enzymatic approach revealed that the UPO of Marasmius rotula is particularly suitable for selective N-dealkylation of the drug and enabled us to prepare both human metabolites via one-pot conversion with an overall yield of 66% N,N-di-desmethyl metabolite and 49% of N-mono-desmethylated compound in two separated kinetic-controlled reactions. KW - Peroxgenase Y1 - 2015 UR - http://www.sciencedirect.com/science/article/pii/S0968089615005295 U6 - https://doi.org/10.1016/j.bmc.2015.06.035 SN - 0968-0896 VL - 23 IS - 15 SP - 4324 EP - 4332 ER - TY - GEN A1 - Ullrich, René A1 - Poraj-Kobielska, Marzena A1 - Scholze, Steffi A1 - Halbout, Claire A1 - Sandvoss, Martin A1 - Pecyna, Marek J. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Side chain removal from corticosteroids by unspecific peroxygenase T2 - Journal of Inorganic Biochemistry N2 - 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, Reichstein's 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 enzyme's 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. KW - Peroxygenation KW - Peroxygenase KW - P450 KW - Deacylation Y1 - 2018 U6 - https://doi.org/10.1016/j.jinorgbio.2018.03.011 SN - 1873-3344 SN - 0162-0134 VL - 183 SP - 84 EP - 93 ER - TY - GEN A1 - Piontek, Klaus A1 - Strittmatter, Eric A1 - Ullrich, René A1 - Gröbe, Glenn A1 - Pecyna, Marek J. A1 - Kluge, Martin A1 - Scheibner, Katrin A1 - Hofrichter, Martin A1 - Plattner, Dietmar A. T1 - Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits T2 - The Journal of Biological Chemistry N2 - Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO. KW - Cytochrome P450 KW - Fungi; Glycoprotein KW - Oxyfunctionalization KW - Polycyclic Aromatic Hydrocarbons KW - Peroxygenase Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.514521 SN - 1083-351X IS - 288 SP - 34767 EP - 34776 ER - 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 - 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 - TY - GEN A1 - Yarman, Aysu A1 - Gröbe, Glenn A1 - Neumann, Bettina A1 - Kinne, Mathias A1 - Gajovic-Eichelmann, Nenad A1 - Wollenberger, Ulla A1 - Hofrichter, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Scheller, Frieder W. T1 - The aromatic peroxygenase from Marasmius rutola—a new enzyme for biosensor applications T2 - Analytical and Bioanalytical Chemistry N2 - The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of −278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe2+/Fe3+ redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed. KW - Unspecific peroxygenase KW - Biosensors KW - Cytochrome P450 KW - Phenolic substances Y1 - 2012 UR - http://link.springer.com/article/10.1007%2Fs00216-011-5497-y U6 - https://doi.org/10.1007/s00216-011-5497-y SN - 1618-2650 VL - 402 IS - 1 SP - 405 EP - 412 ER -