Biochemical Characterization of a Recombinant UDP-glucosyltransferase from Rice and Enzymatic Production of Deoxynivalenol-3-O-β-D-glucoside
Herbert Michlmayr,
Alexandra Malachová,
Elisabeth Varga,
Jana Kleinová,
Marc Lemmens,
Sean Newmister,
Ivan Rayment,
Franz Berthiller,
Gerhard Adam
Affiliations
Herbert Michlmayr
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 24, 3430 Tulln, Austria
Alexandra Malachová
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
Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria
Jana Kleinová
Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria
Marc Lemmens
Biotechnology in Plant Production, Department IFA-Tulln, BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria
Sean Newmister
Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706, USA
Ivan Rayment
Department of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706, USA
Franz Berthiller
Christian Doppler Laboratory for Mycotoxin Metabolism and Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Str. 20, 3430 Tulln, Austria
Gerhard Adam
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 24, 3430 Tulln, Austria
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.
