Structure-guided protein engineering of a novel characterized deoxynivalenol-degrading enzyme from Acidobacteriota provokes enhanced catalytic efficiency

Deoxynivalenol (DON), a typical Fusarium mycotoxin widely distributed in food and feed samples, which poses a global public health risk to humans and animals. Enzymatic detoxification of DON represents a promising approach to control DON contamination. In this work, a novel DON-degrading enzyme (AADH) was characterized from Acidobacteriota with low similarity to other reported DON-degrading enzymes. AADH displayed the highest activity at pH 7.5 and 35 degrees C, and retained >50 % activity after incubation at 40 degrees C for 3 h AADH exhibited great pH stability in the pH range of 5.5-10.0, retaining approximately 60 % residual activities after incubation at 4 degrees C for 12 h Sequentially, structure-guided multiple sequence alignment was applied to enhance the catalytic activity of AADH. In this endeavor, the best mutant E49Q showed a 3.78-fold increase in the specific activity and a 4.06-fold increase in catalytic efficiency (k(cat)/K-m) compared to those of wild-type AADH. Substrate channel analysis, molecular dynamics (MD) simulations, and electrostatic potential analysis suggested that the enhanced catalytic efficiency of E49Q was attributed to the reshaped substrate channel shapes, the shortened attack distance, and the enhanced electrostatic interactions. Accordingly, AADH was generated with improved catalytic efficiency. These results enriched the DON-degrading enzyme libraries, which is of great significance for solving the problem of DON contamination.