Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077
Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-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...
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MDPI AG
2017-02-01
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| Series: | Toxins |
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| Online Access: | http://www.mdpi.com/2072-6651/9/2/58 |
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| author | Herbert Michlmayr Elisabeth Varga Francesca Lupi Alexandra Malachová Christian Hametner Franz Berthiller Gerhard Adam |
| author_facet | Herbert Michlmayr Elisabeth Varga Francesca Lupi Alexandra Malachová Christian Hametner Franz Berthiller Gerhard Adam |
| author_sort | Herbert Michlmayr |
| collection | DOAJ |
| description | Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-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 in Escherichia 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, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-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. Recombinant HvUGT14077 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. |
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| language | English |
| last_indexed | 2024-04-11T22:12:57Z |
| publishDate | 2017-02-01 |
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| series | Toxins |
| spelling | doaj.art-bae893fe3a6743758b63509c744f55ac2022-12-22T04:00:30ZengMDPI AGToxins2072-66512017-02-01925810.3390/toxins9020058toxins9020058Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077Herbert Michlmayr0Elisabeth Varga1Francesca Lupi2Alexandra Malachová3Christian Hametner4Franz Berthiller5Gerhard Adam6Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Straße 24, 3430 Tulln, AustriaCenter for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, AustriaCenter for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, AustriaCenter for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, AustriaInstitute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, AustriaCenter for Analytical Chemistry and Christian Doppler Laboratory for Mycotoxin Metabolism, Department of Agrobiotechnology (IFA-Tulln), BOKU, Konrad Lorenz Straße 20, 3430 Tulln, AustriaDepartment of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Straße 24, 3430 Tulln, AustriaZearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-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 in Escherichia 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, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-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. Recombinant HvUGT14077 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.http://www.mdpi.com/2072-6651/9/2/58zearalenone-14-glucosidezearalenone-16-glucosidemasked mycotoxinglycosylationsucrose synthasesecondary metabolite |
| spellingShingle | Herbert Michlmayr Elisabeth Varga Francesca Lupi Alexandra Malachová Christian Hametner Franz Berthiller Gerhard Adam Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 Toxins zearalenone-14-glucoside zearalenone-16-glucoside masked mycotoxin glycosylation sucrose synthase secondary metabolite |
| title | Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 |
| title_full | Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 |
| title_fullStr | Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 |
| title_full_unstemmed | Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 |
| title_short | Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077 |
| title_sort | synthesis of mono and di glucosides of zearalenone and α β zearalenol by recombinant barley glucosyltransferase hvugt14077 |
| topic | zearalenone-14-glucoside zearalenone-16-glucoside masked mycotoxin glycosylation sucrose synthase secondary metabolite |
| url | http://www.mdpi.com/2072-6651/9/2/58 |
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