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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Mycotoxin Prevention and Applied Microbiology Research » Research » Publications at this Location » Publication #343941

Research Project: Novel Methods for Controlling Trichothecene Contamination of Grain and Improving the Climate Resilience of Food Safety and Security Programs

Location: Mycotoxin Prevention and Applied Microbiology Research

Title: Determinants and expansion of specificity in a trichothecene UDP-glucosyltransferase from Oryza sativa

item WETTERHOWN, KARL - University Of Wisconsin
item GABARDI, KAITLYN - University Of Wisconsin
item MICHLMAYR, HERBERT - University Of Natural Resources & Applied Life Sciences - Austria
item MALACHOVA, ALEXANDRA - University Of Natural Resources & Applied Life Sciences - Austria
item Busman, Mark
item McCormick, Susan
item BERTHILLER, FRANZ - University Of Natural Resources & Applied Life Sciences - Austria
item ADAM, GERHARD - University Of Natural Resources & Applied Life Sciences - Austria
item RAYMENT, IVAN - University Of Wisconsin

Submitted to: Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/15/2017
Publication Date: 11/15/2017
Citation: Wetterhorn, K.M., Gabardi, K., Michlmayr, H., Malachova, A., Busman, M., McCormick, S.P., Berthiller, F., Adam, G., Rayment, I. 2017. Determinants and expansion of specificity in a trichothecene UDP-glucosyltransferase from Oryza sativa. Journal of Biochemistry. 56(50):6585-6596.

Interpretive Summary: In this research we modified a plant glucosyltransferase enzyme that detoxifies the trichothecene mycotoxin deoxynivalenol (DON). DON is produced by Fusarium graminearum, a fungus that causes Fusarium Head Blight (FHB). FHB is a devastating disease of small grain cereal crops that results in both yield reductions and contamination of grain with DON. Trichothecenes are important virulence factors for FHB; therefore, plants that can detoxify trichothecenes have improved resistance to the disease. There is a need for enzymes with broader or altered specificity because a wide range of trichothecenes can be produced by Fusarium species that cause FHB worldwide. In this study, X-ray crystallography was used to guide modification of a glucosyltransferase in rice plants in order to produce mutant enzymes that are able to disable other trichothecenes as well as DON. The mutant enzyme provides the increased specificity required to control FHB across a broad spectrum of Fusarium species and is a good candidate for incorporation in transgenic cereals.

Technical Abstract: Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.