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.

Cereal crops such as wheat, barley, and maize are frequently infected by Fusarium species, causing drastic yield losses and contamination with harmful mycotoxins. Deoxynivalenol (DON) in particular, produced by the widespread species Fusarium graminearum, is a commonly detected mycotoxin in Europe. DON acts as a virulence factor that helps the fungus spread on the host plant, and it is highly toxic to both humans and animals upon ingestion of contaminated cereal products. Investigating the enzymatic detoxification of DON is crucial for elucidating the molecular mechanisms underlying Fusarium resistance in plants and for developing new strategies to reduce DON exposure and its associated risks to humans and livestock. A key plant defense mechanism is detoxification by conjugation - the chemical addition of a water-soluble molecule like glucose or glutathione to a toxic metabolite. Such neutralized toxins (conjugates) are usually stored in the plant tissue. In this project, funded by the Erwin Schrödinger program of the FWF, mechanisms and impact of DON-detoxification by glutathione transferases (GSTs) were studied. While GSTs are well known for their role in herbicide resistance of crop plants, their broader physiological functions in plants are still insufficiently understood. Whether plant GSTs are involved in DON-detoxification was unknown so far and we were the first to demonstrate that wheat GSTs can detoxify DON. During a two-year visit at the University of Turku, Finland, the three-dimensional structures of such GSTs were elucidated by X-ray crystallography and we could show how DON is bound at the active site of such a GST during catalysis. Part of this work was published in the prestigious Journal of Biological Chemistry. Furthermore, we identified highly efficient DON-detoxifying GSTs from different filamentous fungi including Fusarium, Aspergillus, and Trichoderma. These enzymes specifically target the chemical group mainly responsible for the toxicity of DON. The physiological importance of this activity for fungi is so far unknown. Transgenic wheat plants engineered to overexpress such an enzyme from the cellulose degrading fungus Trichoderma reesei showed high resistance to both DON and Fusarium infection. While we detected rapid formation of DON-glutathione conjugates in the transgenic plants, no such compounds were detected in ripened wheat heads. This suggests that these conjugates are completely further metabolized over time. We are currently tracing these unknown downstream metabolites of DON-glutathione conjugates in wheat by isotope-guided untargeted analytics. This project significantly advances our understanding of how plants and fungi are able to neutralize DON. Demonstrating that GSTs can successfully confer Fusarium resistance to wheat provides a new angle for sustainably breeding more resilient crops and thus ensuring both safety and security of our food supply.