TY - GEN A1 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary-like human hepatocytes genetically engineered to obtain proliferation competence display hepatic differentiation characteristics in monolayer and organotypical spheroid cultures T2 - Cell Biology International N2 - Primary human hepatocytes are in great demand during drug development and in hepatology. However, both scarcity of tissue supply and donor variability of primary cells create a need for the development of alternative hepatocyte systems. By using a lentivirus vector system to transfer coding sequences of Upcyte® proliferation genes, we generated non-transformed stable hepatocyte cultures from human liver tissue samples. Here, we show data on newly generated proliferation-competent HepaFH3 cells investigated as conventional two-dimensional monolayer and as organotypical three-dimensional (3D) spheroid culture. In monolayer culture, HepaFH3 cells show typical cobblestone-like hepatocyte morphology and anchorage-dependent growth for at least 20 passages. Immunofluorescence staining revealed that characteristic hepatocyte marker proteins cytokeratin 8, human serum albumin, and cytochrome P450 (CYP) 3A4 were expressed. Quantitative real-time PCR analyses showed that expression levels of analyzed phase I CYP enzymes were at similar levels compared to those of cultured primary human hepatocytes and considerably higher than in the liver carcinoma cell line HepG2. Additionally, transcripts for phase II liver enzymes and transporter proteins OATP-C, MRP2, Oct1, and BSEP were present in HepaFH3. The cells produced urea and converted model compounds such as testosterone, diclofenac, and 7-OH-coumarin into phases I and II metabolites. Interestingly, phases I and II enzymes were expressed at about the same levels in convenient monolayer cultures and complex 3D spheroids. In conclusion, HepaFH3 cells and related primary-like hepatocyte lines seem to be promising tools for in vitro research of liver functions and as test system in drug development and toxicology analysis. KW - cytochrome P450 enzyme Y1 - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbin.10574/full U6 - https://doi.org/10.1002/cbin.10574 SN - 1095-8355 VL - 40 IS - 3 SP - 341 EP - 353 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 - Babot, Esteban D. A1 - Río, José C. del A1 - Cañellas, Marina A1 - Sancho, Ferran A1 - Lucas, Fátima A1 - Guallar, Víctor A1 - Kalum, Lisbeth A1 - Lund, Henrik A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study T2 - Applied and Environmental Microbiology N2 - The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally-friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, and steroid hydrocarbons and ketones were followed by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating (interestingly antiviral and other biological activities of 25-hydroxycholesterol have been recently reported). However, hydroxylation in the ring moiety and terminal hydroxylation at the side-chain was also observed in some steroids, the former favored by the absence of oxygenated groups at C3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active site geometry and hydrophobicity favors the entrance of the steroid side-chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side-chain entrance ratio could be established, that explains the varying reaction yields observed. KW - Peroxyenase Y1 - 2015 UR - http://aem.asm.org/content/early/2015/04/08/AEM.00660-15 U6 - https://doi.org/10.1128/AEM.00660-15 SN - 0099-2240 SN - 1098-5336 VL - 81 IS - 12 SP - 4130 EP - 4142 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Peter, Sebastian A1 - Leonhardt, Sabrina A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Immobilization of unspecific peroxygenases (EC 1.11.2.1) in PVA/PEG gel and hollow fiber modules T2 - Biochemical Engineering Journal N2 - The immobilization of enzymes has many advantages, such as higher stability, easier handling, and reuse of the catalyst. Here we report, for the first time, two effective methods for the immobilization of unspecific peroxygenase (UPO; EC 1.11.2.1). This biocatalyst type comprises heavily glycosylated heme-thiolate proteins that catalyze various biotechnologically relevant oxyfunctionalizations. Both the encapsulation in cryogel and the retention of the enzyme in hollow fiber modules were found to be efficient methods for their immobilization. After encapsulation, the enzyme still exhibited 60% of its initial activity. Interestingly, we did not find differences in the kinetic parameters of free and immobilized UPOs. In long-term experiments, the conversion of the pharmaceutical diclofenac with immobilized UPOs in different reactor types yielded between 62 mg and 154 mg of the major human drug metabolite 4′-hydroxydiclofenac. The maximal total turnover number was about 60-fold higher compared to the free enzyme. A test over 5 months showed that storage of encapsulated UPOs in non-polar solvents (e.g., cyclohexane) helps to preserve the enzyme stability and increases their relative activity (by about ∼150%, in the case of diclofenac hydroxylation). In addition to the hydrophilic substrate diclofenac, encapsulated UPOs also oxidized the hydrophobic model compound cyclohexane. KW - Peroxygenase Y1 - 2015 UR - http://www.sciencedirect.com/science/article/pii/S1369703X15000820 U6 - https://doi.org/10.1016/j.bej.2015.02.037 SN - 1369-703X VL - 98 SP - 144 EP - 150 ER - TY - GEN A1 - Kiebist, Jan A1 - Schmidtke, Kai-Uwe A1 - Zimmermann, Jörg A1 - Kellner, Harald A1 - Jehmlich, Nico A1 - Ullrich, René A1 - Zänder, Daniel A1 - Hofrichter, Martin A1 - Scheibner, Katrin T1 - A peroxygenase from Chaetomium globosum catalyzes the selective oxygenation of testosterone T2 - ChemBioChem N2 - Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates including less activated hydrocarbons by transferring peroxide-borne oxygen. Herein, we investigated a cell-free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule of testosterone by a novel unspecific peroxygenase that was produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass of 36 kDa and specific activities of 4.4 to 12 U mg-1. Whereas well-known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with up to 7,000 total turnovers (TTN) into two oxygenated products: the 4,5-epoxide of testosterone in β-configuration and 16α-hydroxytestosterone. The reaction was performed at 100-mg scale resulting in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, which both could be isolated with purities above 96 %. Thus, CglUPO may be a promising biocatalyst for the oxyfunctionalization of bulky steroids and provide a useful tool for the synthesis of pharmaceutically relevant steroidal molecules. KW - peroxidase KW - hydroxylation Y1 - 2017 UR - http://onlinelibrary.wiley.com/doi/10.1002/cbic.201600677/abstract U6 - https://doi.org/10.1002/cbic.201600677 SN - 1439-7633 VL - 18 IS - 6 SP - 563 EP - 569 ER - TY - GEN A1 - Karich, Alexander A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin T1 - Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction T2 - Journal of Molecular Catalysis B: Enzymatic N2 - The catalase activity of three unspecific peroxygenases (UPOs) from the agaric basidiomycetes Agrocybe aegerita, Coprinopsis cinerea and Marasmius rotula was investigated. The study included analysis of pH dependency of the catalase reaction and H₂O₂ mediated enzyme inactivation as well as experiments on the influence of a second substrate on the course of catalase reaction. Apparent kinetic parameters (Km, kcat) for the catalase activity of UPOs were determined. Inactivation of UPOs by H₂O₂ is discussed with regard to O₂ production and remaining UPO activity. Furthermore formation of biliverdin as heme destruction product was demonstrated along with the formation of UPO compound III as a possible intermediate that forces the destruction process. Radical trapping experiments with methyl benzoate gave indication for the formation of hydroxyl radicals in the presence of excess H₂O₂. Eventually, a plausible pathway of heme destruction has been proposed, proceeding via UPO compound III and subsequent hydroxyl radical formation, which in turn may cause heme bleaching and verdoheme and biliverdin formation. KW - Peroxygenase Y1 - 2016 UR - http://www.sciencedirect.com/science/article/pii/S1381117716302090 U6 - https://doi.org/10.1016/j.molcatb.2016.10.014 SN - 1381-1177 VL - 134 IS - A SP - 238 EP - 246 ER - TY - GEN A1 - Olmedo, Andrés A1 - Aranda, Carmen A1 - Rio, José C. del A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase T2 - Angewandte Chemie International Edition N2 - A new heme–thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position—a challenging reaction in organic chemistry—with H2O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC–MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of 18O from the cosubstrate H218O2, demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids. KW - Peroxyenase Y1 - 2016 UR - http://onlinelibrary.wiley.com/doi/10.1002/anie.201605430/abstract U6 - https://doi.org/10.1002/anie.201605430 SN - 1521-3773 VL - 55 IS - 40 SP - 12248 EP - 12251 ER - TY - GEN A1 - Herzog, Natalie A1 - Hansen, Max A1 - Miethbauer, Sebastian A1 - Schmidtke, Kai-Uwe A1 - Anderer, Ursula A1 - Lupp, Amelie A1 - Sperling, Sebastian A1 - Seehofer, Daniel A1 - Damm, Georg A1 - Scheibner, Katrin A1 - Küpper, Jan-Heiner T1 - Primary like human hepatocytes genetically engineered to obtain proliferation competence display liver biotransformation activity in 2D and 3D culture systems T2 - Cell Biology International Y1 - 2016 SN - 1095-8355 SN - 1065-6995 VL - 40 IS - 3 SP - 341 EP - 353 ER - TY - GEN A1 - Karich, Alexander A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Fungal unspecific peroxygenases oxidize the majority of organic EPA priority pollutants T2 - Frontiers in Microbiology N2 - Unspecific peroxygenases (UPOs) are secreted fungal enzymes with promiscuity for oxygen transfer and oxidation reactions. Functionally, they represent hybrids of P450 monooxygenases and heme peroxidases; phylogenetically they belong to the family of heme-thiolate peroxidases. Two UPOs from the basidiomycetous fungi Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) converted 35 out of 40 compounds listed as EPA priority pollutants, including chlorinated benzenes and their derivatives, halogenated biphenyl ethers, nitroaromatic compounds, polycyclic aromatic hydrocarbons (PAHs) and phthalic acid derivatives. These oxygenations and oxidations resulted in diverse products and-if at all-were limited for three reasons: (i) steric hindrance caused by multiple substitutions or bulkiness of the compound as such (e.g., hexachlorobenzene or large PAHs), (ii) strong inactivation of aromatic rings (e.g., nitrobenzene), and (iii) low water solubility (e.g., complex arenes). The general outcome of our study is that UPOs can be considered as extracellular counterparts of intracellular monooxygenases, both with respect to catalyzed reactions and catalytic versatility. Therefore, they should be taken into consideration as a relevant biocatalytic detoxification and biodegradation tool used by fungi when confronted with toxins, xenobiotics and pollutants in their natural environments. KW - Peroxygenase Y1 - 2017 UR - https://www.frontiersin.org/articles/10.3389/fmicb.2017.01463/full U6 - https://doi.org/10.3389/fmicb.2017.01463 SN - 1664-302X VL - 8 ER - TY - GEN A1 - Olmedo, Andrés A1 - Río, José C. del A1 - Kiebist, Jan A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Fatty Acid Chain Shortening by a Fungal Peroxygenase T2 - Chemistry A European Journal N2 - A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and longchain mono- and dicarboxylic acids by itself alone, in the presence of H₂O₂. The mechanism, analyzed using H₂O, starts with an a-oxidation catalyzed by MroUPO generat- ing an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains. KW - Peroxygenase Y1 - 2017 U6 - https://doi.org/10.1002/chem.201704773 SN - 1521-3765 SN - 0947-6539 VL - 23 SP - 16989 EP - 67 ER - TY - GEN A1 - Martinez, Angel T. A1 - Ruiz-Duenas, Francisco J. A1 - Camarero, Susana A1 - Serrano, Ana A1 - Linde, Dolores A1 - Lund, Henrik A1 - Vind, Jesper A1 - Tovborg, Morton A1 - Herold-Majumdar, Owik M. A1 - Hofrichter, Martin A1 - Liers, Christiane A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Sannia, Giovanni A1 - Piscitelli, Alessandra A1 - Sener, Mehmet E. A1 - Kilic, Sibel A1 - Berkel, Willem J. H. van A1 - Guallar, Víctor A1 - Lucas, Maria Fátima A1 - Zuhse, Ralf A1 - Ludwig, Roland A1 - Hollmann, Frank A1 - Fernández-Fueyo, Elena A1 - Record, Eric A1 - Faulds, Craig B. A1 - Tortajada, Marta A1 - Winckelmann, Ib A1 - Rasmussen, Jo-Anne A1 - Gelo-Pujic, Mirjana A1 - Gutiérrez, Ana A1 - Rio, José C. del A1 - Rencoret, Jorge A1 - Alcalde, Miguel T1 - Oxidoreductases on their way to industrial biotransformations T2 - Biotechnology Advances N2 - Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations. KW - Peroxygenase KW - Biotechnology Y1 - 2017 U6 - https://doi.org/10.1016/j.biotechadv.2017.06.003 SN - 1873-1899 SN - 0734-9750 VL - 35 IS - 6 SP - 815 EP - 831 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 - Steinbrecht, Susanne A1 - König, Rosalie A1 - Schmidtke, Kai-Uwe A1 - Herzog, Natalie A1 - Scheibner, Katrin A1 - Krüger-Genge, Anne A1 - Jung, Friedrich A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner T1 - Metabolic activity testing can underestimate acute drug cytotoxicity as revealed by HepG2 cell clones overexpressing cytochrome P450 2C19 and 3A4 T2 - Toxicology Y1 - 2018 U6 - https://doi.org/10.1016/j.tox.2018.11.008 SN - 0300-483X VL - 412 SP - 37 EP - 47 ER - TY - GEN A1 - Aranda, Carmen A1 - Ullrich, René A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Río, José C. del A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases T2 - Catalysis Science & Technology N2 - This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000) than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV. KW - Peroxygenase Y1 - 2018 U6 - https://doi.org/10.1039/C8CY00272J SN - 2044-4761 SN - 2044-4753 VL - 9 IS - 8 SP - 2394 EP - 2401 ER - TY - GEN A1 - Aranda, Carmen A1 - Olmedo, Andrés A1 - Kiebist, Jan A1 - Scheibner, Katrin A1 - Río, José C. del A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters by Fungal Peroxygenases T2 - CHEMCATCHEM N2 - Recently discovered fungal unspecific peroxygenases from Marasmius rotula and Chaetomium globosum catalyze the epoxidation of unsaturated fatty acids (FA) and FA methyl esters (FAME), unlike the well‐known peroxygenases from Agrocybe aegerita and Coprinopsis cinerea. Reactions of a series of unsaturated FA and FAME with cis‐configuration revealed high (up to 100 %) substrate conversion and selectivity towards epoxidation, although some significant differences were observed between enzymes and substrates with the best results being obtained with the C. globosum enzyme. This and the M. rotula peroxygenase appear as promising biocatalysts for the environmentally‐friendly production of reactive FA epoxides given their self‐sufficient monooxygenase activity and the high conversion rate and epoxidation selectivity. KW - Peroxygenase Y1 - 2018 U6 - https://doi.org/10.1002/cctc.201800849 SN - 1867-3899 VL - 10 IS - 18 SP - 3964 EP - 3968 ER - TY - GEN A1 - Steinbrecht, Susanne A1 - Kiebist, Jan A1 - König, Rosalie A1 - Thiessen, Markus A1 - Schmidtke, Kai-Uwe A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Scheibner, Katrin T1 - Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells T2 - AMB Express N2 - Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32% (purity > 97.6%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO–induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment. KW - Biocatalysis KW - Cyclophosphamide KW - Human drug metabolites KW - Peroxygenase KW - Toxicity Y1 - 2020 UR - https://amb-express.springeropen.com/articles/10.1186/s13568-020-01064-w U6 - https://doi.org/10.1186/s13568-020-01064-w SN - 2191-0855 VL - 10 ER - TY - GEN A1 - Ingenbosch, Kim N. A1 - Quint, Stephan A1 - Dyllick-Brenzinger, Melanie A1 - Wunschik, Dennis S. A1 - Kiebist, Jan A1 - Süss, Philipp A1 - Liebelt, Ute A1 - Zuhse, Ralf A1 - Menyes, Ulf A1 - Scheibner, Katrin A1 - Mayer, Christian A1 - Opwis, Klaus A1 - Gutmann, Jochen S. A1 - Hoffmann-Jacobsen, Kerstin T1 - Singlet oxygen generation by peroxidases and peroxygenases for chemo-enzymatic synthesis T2 - ChemBioChem N2 - Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singlet‐oxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet‐oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet‐oxygen formation. All peroxidases and peroxygenases show singlet‐oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye‐decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet‐oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen. KW - Peroxygenase Y1 - 2021 U6 - https://doi.org/10.1002/cbic.202000326 SN - 1439-7633 SN - 1439-4227 VL - 22 IS - 2 SP - 398 EP - 407 ER - TY - CHAP A1 - Hofrichter, Martin A1 - Kellner, Harald A1 - Herzog, Robert A1 - Karich, Alexander A1 - Liers, Christiane A1 - Scheibner, Katrin A1 - Kimani, Virginia Wambui A1 - Ullrich, René ED - Nevalainen, Helena T1 - Fungal Peroxygenases: A Phylogenetically Old Superfamily of Heme Enzymes with Promiscuity for Oxygen Transfer Reactions. T2 - Grand Challenges in Fungal Biotechnology KW - Peroxygenase Y1 - 2020 SN - 978-3-030-29540-0 SN - 978-3-030-29541-7 U6 - https://doi.org/10.1007/978-3-030-29541-7 SN - 2367-1017 SN - 2367-1025 SP - 369 EP - 403 PB - Springer Nature CY - Cham ET - 1. Auflage ER - TY - CHAP A1 - Kiebist, Jan A1 - Hofrichter, Martin A1 - Zuhse, Ralf A1 - Scheibner, Katrin ED - Grunwald, Peter T1 - Oxyfunctionalization of Pharmaceuticals by Fungal Peroxygenases T2 - Pharmaceutical biocatalysis : chemoenzymatic synthesis of active pharmaceutical ingredients N2 - Throughout drug discovery and development, metabolic studies are driven by an increased interest to understand the potential for side effects and drug-drug interactions. Peroxygenases are a subclass of peroxide-dependent enzymes that catalyze the transfer of a peroxide-borne oxygen to diverse substrates. The selective oxyfunctionalization of organic molecules is one of the major challenges for the chemical community. Benzylic hydroxylation is one of the most frequently observed reactions of unspecific peroxygenases due to the activated nature of benzylic C–H bonds. The hydroxylation of aromatic rings is a common reaction in the formation of drug metabolites by P450s in mammals including humans. In the liver, P450s facilely metabolize secondary and tertiary amines as well as ethers to the corresponding dealkylated metabolites. The regio- and stereoselective direct introduction of oxygen functionalities into complex pharmaceuticals is a great challenge for organic chemists. KW - Peroxygenase Y1 - 2019 SN - 978-981-4800-80-8 SN - 978-1-00-070757-1 SP - 643 EP - 673 PB - Jenny Stanford Publishing Pte. Ltd. CY - Singapore ET - 1. Auflage ER - TY - GEN A1 - Kiebist, Jan A1 - Koncz, Tino A1 - Friedrich, Stephanie A1 - Scheibner, Katrin T1 - Oxidative biocatalysts to design new oxyfunctionalization tools for drugs and added value bio-based products T2 - International Biotech Innovation Days 2020 (IBID), 28th – 29th October 2020 KW - Peroxygenase Y1 - 2020 UR - https://www.b-tu.de/ibid/program#c243720 CY - Senftenberg ER -