Deoxynivalenol (DON) is a toxic fungal secondary metabolite produced by Fusarium graminearum which causes Fusarium Head Blight and Pink Ear Rot disease in wheat and corn respectively. DON is a predominant contaminant in cereal grain crops with outbreaks costing the North American cereal grain industry millions of dollars annually. There is a growing need for effective DON mitigation strategies due to DON’s inherent toxicity which affects the performance of livestock fed contaminated grain.
Current DON management strategies involve physical decontamination or marginally effective chemical treatments; however, a holistic and targeted approach via the incorporation of DON detoxifying enzymes is a promising strategy. Previous studies demonstrated that D. mutans 17-2-E-8, a soil bacterium, epimerizes DON to the less toxic 3-epi-DON via the intermediate, 3-keto-DON. The process involves two enzymes, DepA, a PQQ-dependent dehydrogenase, and DepB, an NADPH-dependent aldo-keto reductase (AKR). The strict requirement for the expensive cofactor, NADPH, poses a significant impediment to the practical application of these enzymes. Protein engineering approaches can address this issue – by ‘switching’ DepB’s cofactor preference to the cheaper co-factor, NADH. DepB was found to catalyze the transformation of 3-keto DON to 3-epi DON with Km and kcat values of 563.9 µM and 2.49s-1, respectively, using NADPH as a cofactor. Secondly, the enzyme’s Kd for NADPH was determined to be 44.23 µM using fluorescence enhancement assays. Using the solved crystal structure of DepB, docking experiments with DepB revealed that Arg-289, Gln-293, and Lys-216 may be important for NADPH specificity. Therefore, site-specific mutagenesis was performed to replace these residues to enable the enzyme to utilize NADH. The catalytic efficiencies for these designed mutants will next be determined and compared to catalytic efficiencies of the wild type DepB.
