Detoxification of Deoxynivalenol by Glutathione transferases

Fungi of the genus Fusarium represent a significant challenge in global cereal production as they cause crop losses and contamination with mycotoxins. Deoxynivalenol (DON) produced by the widespread species Fusarium graminearum is a frequently occurring mycotoxin in Europe. This toxin facilitates spread of the fungus on the host plant and is also highly toxic to humans and animals after ingestion of cereal products. Since DON is chemically very stable and not destroyed by heating, strategies for its detoxification are of great interest. In the already completed FWF project SFB Fusarium a major focus of our working group was on the isolation of mainly plant-derived enzymes with the ability to detoxify DON. While most of the enzymes characterized so far catalyze reversible reactions, we were able to identify glutathione transferases that irreversibly detoxify DON. These enzymes permanently eliminate its epoxide group, which is mainly responsible for the toxicity, by binding to glutathione. While the plant glutathione transferases investigated so far catalyze this epoxide opening reaction relatively slowly, we were recently able to identify some highly efficient fungal glutathione transferases. The aim of this project is to elucidate the reaction mechanisms of such glutathione transferases by X- ray crystallography. The enzymes in question will be produced in the host organism Escherichia coli, purified by affinity chromatography, and then crystallized together with DON or the detoxified reaction product DON-glutathione. The structures of these complexes are resolved by X-ray diffraction. Based on this, it can be deduced how DON is positioned at the active site during catalysis and which amino acids are involved in the reaction. The catalytic function of specific amino acids will be tested by replacing them by site directed mutagenesis and evaluating the functionality of the modified enzymes. The work, financed by the Schrödinger program of the FWF, will be carried out over a period of two years at the University of Turku, Finland. In the one-year return phase at BOKU, the enzymes involved in this study will be kinetically characterized. The planned work should significantly increase our understanding of the biocatalytic detoxification of DON and chemically closely related trichothecene-class toxins. The biochemical characterization of the glutathione transferases involved should also lead to new insights into their roles in plants and fungi, and could provide information on improving Fusarium resistance in crops through targeted breeding or genome editing.