Michlmayr Herbert, Varga Elisabeth, Lupi Francesca, Malachová Alexandra, Hametner Christian, Berthiller Franz, Adam Gerhard
Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Straße 24, 3430 Tulln, Austria.
Department of Food Chemistry and Toxicology, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria.
Toxins (Basel). 2017 Feb 9;9(2):58. doi: 10.3390/toxins9020058.
Zearalenone (ZEN) is an estrogenic mycotoxin occurring in -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. UGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in , affinity purified, and characterized. UGT14077 possesses high affinity ( = 3 µM) and catalytic efficiency (/ = 190 s·mM) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, with / of 40 and 74 s·mM, respectively. UGT14077 catalyzes -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 UGT14077 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.
玉米赤霉烯酮（ZEN）是一种存在于受感染谷物中的具有雌激素活性的霉菌毒素。糖基化是一种重要的植物防御机制，通常会降低霉菌毒素对人类和动物的急性毒性。由于缺乏动物研究所需的大量ZEN-葡糖苷，关于它们的毒理学信息有限。UGT14077是一种最近得到验证的能与ZEN结合的大麦UDP-葡糖基转移酶，在（此处原文缺失具体表达宿主）中表达、亲和纯化并进行了表征。UGT14077对ZEN具有高亲和力（Km = 3 µM）和催化效率（Vmax/Km = 190 s·mM）。它还能有效地将I相ZEN代谢物α-玉米赤霉烯醇和β-玉米赤霉烯醇糖基化，其Vmax/Km分别为40和74 s·mM。UGT14077催化在C-14和C-16处的β-糖基化，优先合成14-葡糖苷。此外，观察到14,16-二葡糖苷的相对缓慢的连续形成；它们的结构通过质谱初步鉴定，对于ZEN-14,16-二葡糖苷通过核磁共振光谱得到确认。重组UGT14077实现了ZEN-葡糖苷的高效制备合成，产生约90%的ZEN-14-葡糖苷和10%的ZEN-16-葡糖苷。通过与对ZEN-14-葡糖苷具有高度选择性的β-葡糖苷酶共孵育，ZEN-16-葡糖苷的产量可提高到85%。基于蔗糖合酶的再生系统抵消了共底物UDP-葡萄糖的消耗。该策略对于提高其他葡糖基转移酶合成的次要葡糖苷的产量也可能具有重要意义。
