Improving the Catalytic Activity of a Quinone-Dependent Dehydrogenase for Deoxynivalenol Detoxification by Engineering the Conformation of the N-Terminal Loop.

Abstract

The low oxidation activity of pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DepA) toward deoxynivalenol (DON) restricts its practical application in the food industry. Here, bioinformatics analysis indicated that the N-terminal loop of DepA acts as a lid that swings above the active pocket and plays an important role in controlling substrate entry. Considering that different PQQ-dependent dehydrogenases have different N-terminal loop lengths, we constructed DepA N-terminal truncation mutants. The catalytic efficiency (kcat/Km) of the best truncation mutant, N21, increased by 7.9-fold. Computational analysis suggested that mutant N21 maintains the open conformation of the N-terminal loop and widens the substrate channel to increase the level of substrate entry. In addition, the active pocket of mutant N21 was reshaped, which strengthened the interaction between DON and the active cavity residues and reduced the proton transfer distance, enhancing catalytic activity. This study provides new insights into engineering the catalytic activity of PQQ-dependent dehydrogenases.