title: Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077.
abstract: Zearalenone (ZEN) is an estrogenic mycotoxin occurring inFusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies.HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed inEscherichia coli, affinity purified, and characterized.HvUGT14077 possesses high affinity (Km= 3 µM) and catalytic efficiency (kcat/Km= 190 s-1·mM-1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, withkcat/Kmof 40 and 74 s-1·mM-1, respectively.HvUGT14077 catalyzesO-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. RecombinantHvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a β-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Straße 24, 3430 Tulln, Austria. herbert.michlmayr@univie.ac.at.|Department of Food Chemistry and Toxicology, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria. herbert.michlmayr@univie.ac.at.
authors.forename: Herbert
authors.initials: H
authors.lastname: Michlmayr
authors.affiliation: Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, Austria. elisabeth.varga@boku.ac.at.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Varga
authors.affiliation: Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, Austria. francesca.lupi@unifg.it.|Dipartimento di Scienze Agrarie, degli Alimenti e dell'Ambiente, Università degli Studi di Foggia, Via-Napoli 25, 71122 Foggia, Italy. francesca.lupi@unifg.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Lupi
authors.affiliation: Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, Austria. alexandra.malachova@boku.ac.at.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Malachová
authors.affiliation: Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, Austria. christian.hametner@tuwien.ac.at.
authors.forename: Christian
authors.initials: C
authors.lastname: Hametner
authors.affiliation: Center for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, Austria. franz.berthiller@boku.ac.at.
authors.forename: Franz
authors.initials: F
authors.lastname: Berthiller
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Straße 24, 3430 Tulln, Austria. gerhard.adam@boku.ac.at.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Adam
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005960:Glucosides / Q000737:chemistry / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology; D015394:Molecular Structure; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015025:Zearalenone / Q000378:metabolism*
chemical_list: D005960:Glucosides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D015025:Zearalenone; D005964:Glucosyltransferases
keywords: glycosylation; masked mycotoxin; secondary metabolite; sucrose synthase; zearalenone-14-glucoside; zearalenone-16-glucoside