Deoxynivalenol (DON) and its acetylated derivatives such as 3-acetyldeoxynivalenol (3A-DON) and 15-acetyldeoxynivalenol (15A-DON) are notorious mycotoxins in Fusarium contaminated cereals, which pose a great threat to human and livestock health. The specialized glyoxalase I from Gossypium hirsutum (SPG) can lower the toxicity of 3A-DON by conducting isomerization to transfer C8 carbonyl to C7 and double bond from C9-C10 to C8-C9. Here we report that the substrate-flexible SPG can also recognize 15A-DON and DON, probably following the same isomerization mechanism as that for 3A-DON. The crystallographic, mutagenesis, and biochemical analyses revealed that SPG provides a hydrophobic pocket to accommodate the substrate and residue E167 might serve as the catalytic base. A variant SPGY62A that was constructed based on structure-based protein engineering exhibited elevated catalytic activity towards DON, 3A-DON, and 15A-DON by >70%. Furthermore, variant SPGY62A was successfully expressed in Pichia pastoris, whose catalytic activity was also compared to that produced in Escherichia coli. These results provide a blueprint for further protein engineering of SPG and reveal the potential applications of the enzyme in detoxifying DON, 3A-DON and 15A-DON.
Keywords: Crystal structure; Deoxynivalenol; Pichia pastoris; Protein engineering; Specialized glyoxalase I.
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