Identification of a novel DON dehydrogenase and computational insights into the decisive role of cofactor affinity in DON detoxification

Deoxynivalenol (DON) is a pervasive mycotoxin contaminating cereal crops, thereby posing significant risks to both human and animal health. A novel DON dehydrogenase, KvDDH, was identified as an efficient catalyst for degrading DON. The enzyme exhibited optimal activity at 35 °C and pH 6.0, in the presence of preferred cofactor phenazine methosulfate (PMS). Computational approaches integrated AI-driven structural modeling, molecular docking, density functional theory (DFT) calculations, and molecular dynamics (MD) simulation to reveal the affinity between enzyme and cofactors. DFT-based molecular surface electrostatic potential analysis revealed that the nucleophilic sites of PMS, 2,6-dichlorophenolindophenol (DCPIP), and pyrroloquinoline quinone (PQQ) are strategically positioned to accept protons, thereby facilitating protonation. Notably, MD simulations demonstrated that the proton-accepting group of PMS maintains the shortest average distance (5.88 Å) to the catalytic residue Asp306 in KvDDH, accompanied by the lowest binding free energy (ΔG_bind = −28.67 kcal/mol) among the cofactors tested. Furthermore, degradation product analysis confirmed the conversion of DON to 3-keto-DON, supporting a catalytic mechanism that involves cyclic regeneration of PMS and DCPIP. Asp306 appears to mediate proton transfer from the C3-hydroxyl group of DON to the cofactors, thereby promoting electron transfer and cofactor regeneration during dehydrogenation. These findings underscore the utility of computational strategies for accurately predicting cofactor-DON dehydrogenase interactions.