1st Place: Structural and functional characterization of a delta-class glutathione S-transferase in the European honey bee, Apis mellifera

The European honey bee, Apis mellifera, is an essential pollinator species managed by humankind for thousands of years. Despite their ecological and economic importance, several studies have shown that honey bees are facing serious health decline from combined biotic and abiotic stress including diseases, limited nutrition, and agrochemicals. There is a very limited understanding of the detoxification mechanism of toxic chemical substances in pollinators, especially the Glutathione-S Transferase (GSTs), a widely studied class of enzyme known for its vital role in detoxification and a protective role against oxidative stress. In this study, we structurally and functionally characterized a delta subclass GST enzyme, AmGSTD1, with its roles in agrochemical detoxification and antioxidative stress. Quantitative real-time PCR revealed relatively high expression of AmGSTD1 in malpighian tubules compared to other tissues in both nurse and forage bees. The protective role of recombinant AmGSTD1 against known oxidative stress inducers (hydrogen peroxide and cumene hydroperoxide) was observed via disc diffusion assay. The three-dimensional structure of AmGSTD1 protein was solved by X-ray crystallography to identify its active site. The enzymatic assay was performed to determine enzyme activity and kinetic parameters using 1-chloro-2,4-dinitrobenzene, glutathione, and potential pesticides found in agricultural fields as substrates. Mutants of AmGSTD1 were constructed with site-directed mutagenesis in both G and H binding sites of the active site and the activities of mutants were investigated with enzymatic assay. The results indicate that AmGSTD1 plays important role in detoxifying agrochemicals and reducing oxidative stress, which serves as a key enzyme for bee survival.