@misc{SteinbrechtKiebistKoenigetal., author = {Steinbrecht, Susanne and Kiebist, Jan and K{\"o}nig, Rosalie and Thiessen, Markus and Schmidtke, Kai-Uwe and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Scheibner, Katrin}, title = {Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells}, series = {AMB Express}, volume = {10}, journal = {AMB Express}, issn = {2191-0855}, doi = {10.1186/s13568-020-01064-w}, pages = {13}, abstract = {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.}, language = {en} } @misc{ScheibnerKiebistSchmidtkeetal., author = {Scheibner, Katrin and Kiebist, Jan and Schmidtke, Kai-Uwe and K{\"u}pper, Jan-Heiner}, title = {Verfahren zur enzymatischen Herstellung von zytostatischen 4-Hydroxy-Oxazaphosphorinen"}, language = {de} } @misc{KiebistSchmidtkeZimmermannetal., author = {Kiebist, Jan and Schmidtke, Kai-Uwe and Zimmermann, J{\"o}rg and Kellner, Harald and Jehmlich, Nico and Ullrich, Ren{\´e} and Z{\"a}nder, Daniel and Hofrichter, Martin and Scheibner, Katrin}, title = {A peroxygenase from Chaetomium globosum catalyzes the selective oxygenation of testosterone}, series = {ChemBioChem}, volume = {18}, journal = {ChemBioChem}, number = {6}, issn = {1439-7633}, doi = {10.1002/cbic.201600677}, pages = {563 -- 569}, abstract = {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.}, 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{KoenigKiebistKalmbachetal., author = {K{\"o}nig, Rosalie and Kiebist, Jan and Kalmbach, Johannes and Herzog, Robert and Schmidtke, Kai-Uwe and Kellner, Harald and Ullrich, Ren{\´e} and Jehmlich, Nico and Hofrichter, Martin and Scheibner, Katrin}, title = {Novel unspecific peroxygenase from Truncatella angustata catalyzes the synthesis of bioactive lipid mediators}, series = {Microorganisms}, volume = {10}, journal = {Microorganisms}, number = {7}, issn = {2076-2607}, doi = {10.3390/microorganisms10071267}, pages = {1 -- 18}, abstract = {Lipid mediators, such as epoxidized or hydroxylated eicosanoids (EETs, HETEs) of arachidonic acid (AA), are important signaling molecules and play diverse roles at different physiological and pathophysiological levels. The EETs and HETEs formed by the cytochrome P450 enzymes are still not fully explored, but show interesting anti-inflammatory properties, which make them attractive as potential therapeutic target or even as therapeutic agents. Conventional methods of chemical synthesis require several steps and complex separation techniques and lead only to low yields. Using the newly discovered unspecific peroxygenase TanUPO from the ascomycetous fungus Truncatella angustata, 90\% regioselective conversion of AA to 14,15-EET could be achieved. Selective conversion of AA to 18-HETE, 19-HETE as well as to 11,12-EET and 14,15-EET was also demonstrated with known peroxygenases, i.e., AaeUPO, CraUPO, MroUPO, MweUPO and CglUPO. The metabolites were confirmed by HPLC-ELSD, MS1 and MS2 spectrometry as well as by comparing their analytical data with authentic standards. Protein structure simulations of TanUPO provided insights into its substrate access channel and give an explanation for the selective oxyfunctionalization of AA. The present study expands the scope of UPOs as they can now be used for selective syntheses of AA metabolites that serve as reference material for diagnostics, for structure-function elucidation as well as for therapeutic and pharmacological purposes}, language = {en} } @misc{KellnerFriedrichSchmidtkeetal., author = {Kellner, Harald and Friedrich, Stephanie and Schmidtke, Kai-Uwe and Ullrich, Ren{\´e} and Kiebist, Jan and Z{\"a}nder, Daniel and Hofrichter, Martin and Scheibner, Katrin}, title = {Draft genome sequence of Truncatella angustata (Anamorph) S358}, series = {Microbiology resource announcement}, volume = {11}, journal = {Microbiology resource announcement}, number = {7}, issn = {2169-8287}, doi = {10.1128/mra.00052-22}, abstract = {The ascomycete Truncatella angustata has a worldwide distribution. Commonly, it is associated with plants as an endophyte, pathogen, or saprotroph. The genome assembly comprises 44.9 Mbp, a G+C content of 49.2\%, and 12,353 predicted genes, among them 12 unspecific peroxygenases (EC 1.11.2.1).}, language = {en} } @misc{FriedrichSchrammKiebistetal., author = {Friedrich, Stephanie and Schramm, Marina and Kiebist, Jan and Schmidtke, Kai-Uwe and Scheibner, Katrin}, title = {Development of translationally active cell lysates from different filamentous fungi for application in cell-free protein synthesis}, series = {Enzyme and microbial technology}, volume = {185}, journal = {Enzyme and microbial technology}, publisher = {Elsevier BV}, address = {Amsterdam}, doi = {10.1016/j.enzmictec.2025.110588}, pages = {1 -- 10}, abstract = {There is an enormous potential for cell-free protein synthesis (CFPS) systems based on filamentous fungi in view of their simple, fast and mostly inexpensive cultivation with high biomass space-time yields and in view of their catalytic capacity. In 12 of the 22 different filamentous fungi examined, in vitro translation of at least one of the two reporter proteins GFP and firefly luciferase was detected. The lysates showing translation of a reporter protein usually were able to synthesize a functional cell-free expressed unspecific peroxygenase (UPO) from the basidiomycete Cyclocybe (Agrocybe) aegerita. For the most promising candidate Neurospora crassa, the influence of different conditions of cultivation and lysate preparation on in vitro translation of the reporter proteins was investigated and optimized. In general, the greatest improvements in the translational activity were achieved by the choice of the growth medium, the addition of organic nitrogen being most beneficial. Optimizing the culture and preparation conditions of the N. crassa platform improved protein yield of the original lysate by a factor of 25 for firefly luciferase and 17 for GFP, respectively. In addition to the reporter proteins, the aforementioned UPO as well as a functional UPO from Aspergillus niger were cell-free expressed using the different lysates from N. crassa. CFPS with fungal lysates opens the door to expressing UPOs in high throughput and in parallel, for example to optimize synthesis conditions or adapt catalyst properties. The presented method proves the general potential of fungal lysates for application in cell-free syntheses.}, language = {en} } @misc{SchrammCarrilloAvilesKalmbachetal., author = {Schramm, Marina and Carrillo Avil{\´e}s, Carlos Renato and Kalmbach, Johannes and Schmidtke, Kai-Uwe and Kiebist, Jan and Kellner, Harald and Hofrichter, Martin and Scheibner, Katrin}, title = {Rapid screening system to identify unspecific peroxygenase activity}, series = {Clinical hemorheology and microcirculation}, volume = {89}, journal = {Clinical hemorheology and microcirculation}, number = {4}, publisher = {Sage}, address = {London}, issn = {1875-8622}, doi = {10.1177/13860291241306566}, pages = {363 -- 373}, abstract = {Unspecific peroxygenases (UPO, EC 1.11.2.1) are a valuable tool for the biocatalytic synthesis of specialty chemicals such as pharmaceutical metabolites. However, the search for new UPOs that are recombinantly expressible can be tedious and dependent on expensive equipment, especially when a large number of clones has to be examined. In this study, we present a simple agar plate-based method for the screening of active, secreted UPOs heterologously expressed in Saccharomyces cerevisiae. This allows a real high-throughput of several thousand clones at once. The approach was successfully tested with a small gene library comprising putative UPO genes and resulted in the identification of two clones producing short UPOs from the filamentous fungi Dendrothele bispora (DbiUPO) and Aspergillus niger (AniUPO). Both UPOs were partly purified and characterized with respect to their catalytic properties. With differing efficiencies and product specificities, they catalyzed the formation of human drug metabolites, e.g., lipid mediators from polyunsaturated fatty acids and the active metabolite of the prodrug clopidogrel, respectively.}, language = {en} }