@misc{MartinezRuizDuenasCamareroetal., author = {Martinez, Angel T. and Ruiz-Duenas, Francisco J. and Camarero, Susana and Serrano, Ana and Linde, Dolores and Lund, Henrik and Vind, Jesper and Tovborg, Morton and Herold-Majumdar, Owik M. and Hofrichter, Martin and Liers, Christiane and Ullrich, Ren{\´e} and Scheibner, Katrin and Sannia, Giovanni and Piscitelli, Alessandra and Sener, Mehmet E. and Kilic, Sibel and Berkel, Willem J. H. van and Guallar, V{\´i}ctor and Lucas, Maria F{\´a}tima and Zuhse, Ralf and Ludwig, Roland and Hollmann, Frank and Fern{\´a}ndez-Fueyo, Elena and Record, Eric and Faulds, Craig B. and Tortajada, Marta and Winckelmann, Ib and Rasmussen, Jo-Anne and Gelo-Pujic, Mirjana and Guti{\´e}rrez, Ana and Rio, Jos{\´e} C. del and Rencoret, Jorge and Alcalde, Miguel}, title = {Oxidoreductases on their way to industrial biotransformations}, series = {Biotechnology Advances}, volume = {35}, journal = {Biotechnology Advances}, number = {6}, issn = {1873-1899}, doi = {10.1016/j.biotechadv.2017.06.003}, pages = {815 -- 831}, abstract = {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.}, language = {en} } @misc{KiebistSchmidtkeSchrammetal., author = {Kiebist, Jan and Schmidtke, Kai-Uwe and Schramm, Marina and Hofrichter, Martin and K{\"o}nig, Rosalie and Quint, Stephan and Kohlmann, Johannes and Zuhse, Ralf and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin}, title = {Biocatalytic syntheses of antiplatelet metabolites of the thienopyridines clopidogrel and prasugrel using fungal peroxygenases}, series = {Journal of Fungi}, volume = {7}, journal = {Journal of Fungi}, number = {9}, issn = {2309-608X}, doi = {10.3390/jof7090752}, pages = {1 -- 17}, abstract = {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.}, language = {en} } @misc{IngenboschQuintDyllickBrenzingeretal., author = {Ingenbosch, Kim N. and Quint, Stephan and Dyllick-Brenzinger, Melanie and Wunschik, Dennis S. and Kiebist, Jan and S{\"u}ss, Philipp and Liebelt, Ute and Zuhse, Ralf and Menyes, Ulf and Scheibner, Katrin and Mayer, Christian and Opwis, Klaus and Gutmann, Jochen S. and Hoffmann-Jacobsen, Kerstin}, title = {Singlet oxygen generation by peroxidases and peroxygenases for chemo-enzymatic synthesis}, series = {ChemBioChem}, volume = {22}, journal = {ChemBioChem}, number = {2}, issn = {1439-7633}, doi = {10.1002/cbic.202000326}, pages = {398 -- 407}, abstract = {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.}, language = {en} } @incollection{KiebistHofrichterZuhseetal., author = {Kiebist, Jan and Hofrichter, Martin and Zuhse, Ralf and Scheibner, Katrin}, title = {Oxyfunctionalization of Pharmaceuticals by Fungal Peroxygenases}, series = {Pharmaceutical biocatalysis : chemoenzymatic synthesis of active pharmaceutical ingredients}, booktitle = {Pharmaceutical biocatalysis : chemoenzymatic synthesis of active pharmaceutical ingredients}, editor = {Grunwald, Peter}, edition = {1. Auflage}, publisher = {Jenny Stanford Publishing Pte. Ltd.}, address = {Singapore}, isbn = {978-981-4800-80-8}, pages = {643 -- 673}, abstract = {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.}, language = {en} }