title: Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-D-glucoside.
abstract: Glycosylation is an important plant defense mechanism and conjugates of Fusarium mycotoxins often co-occur with their parent compounds in cereal-based food and feed. In case of deoxynivalenol (DON), deoxynivalenol-3-O-β-D-glucoside (D3G) is the most important masked mycotoxin. The toxicological significance of D3G is not yet fully understood so that it is crucial to obtain this compound in pure and sufficient quantities for toxicological risk assessment and for use as an analytical standard. The aim of this study was the biochemical characterization of a DON-inactivating UDP-glucosyltransferase from rice (OsUGT79) and to investigate its suitability for preparative D3G synthesis. Apparent Michaelis constants (Km) of recombinant OsUGT79 were 0.23 mM DON and 2.2 mM UDP-glucose. Substrate inhibition occurred at DON concentrations above 2 mM (Ki = 24 mM DON), and UDP strongly inhibited the enzyme. Cu2+ and Zn2+ (1 mM) inhibited the enzyme completely. Sucrose synthase AtSUS1 was employed to regenerate UDP-glucose during the glucosylation reaction. With this approach, optimal conversion rates can be obtained at limited concentrations of the costly co-factor UDP-glucose. D3G can now be synthesized in sufficient quantity and purity. Similar strategies may be of interest to produce β-glucosides of other toxins.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 24, 3430 Tulln, Austria. herbert.michlmayr@boku.ac.at.
authors.forename: Herbert
authors.initials: H
authors.lastname: Michlmayr
authors.affiliation: Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria. alexandra.malachova@boku.ac.at.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Malachová
authors.affiliation: Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria. elisabeth.varga@boku.ac.at.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Varga
authors.affiliation: Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria. kleinovaja@seznam.cz.|Department of Chemistry and Biochemistry, Mendel University in Brno, Zemědělská 1, 61300 Brno, Czech Republic. kleinovaja@seznam.cz.
authors.forename: Jana
authors.initials: J
authors.lastname: Kleinová
authors.affiliation: Biotechnology in Plant Production, Department IFA-Tulln, BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria. marc.lemmens@boku.ac.at.
authors.forename: Marc
authors.initials: M
authors.lastname: Lemmens
authors.affiliation: Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706, USA. snewmist@umich.edu.
authors.forename: Sean
authors.initials: S
authors.lastname: Newmister
authors.affiliation: Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706, USA. ivan_rayment@biochem.wisc.edu.
authors.forename: Ivan
authors.initials: I
authors.lastname: Rayment
authors.affiliation: Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 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 Str. 24, 3430 Tulln, Austria. gerhard.adam@boku.ac.at.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Adam
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005960:Glucosides / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D012275:Oryza / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D011994:Recombinant Proteins; D053719:Tandem Mass Spectrometry; D014255:Trichothecenes / Q000378:metabolism* / Q000633:toxicity
chemical_list: D005960:Glucosides; D011994:Recombinant Proteins; D014255:Trichothecenes; C542050:deoxynivalenol-3-glucoside; D005964:Glucosyltransferases; C007262:deoxynivalenol
keywords: Fusarium; UDP-glucose recycling; glycosylation; masked mycotoxin; sucrose synthase