Loops typically allosterically communicate with active sites, and their conformational dynamics can affect the catalytic properties of enzymes. Herein, by manipulating the loop conformational dynamics via adjusting loop-loop interactions, a medium-chain alcohol dehydrogenase (Syn94) was engineered to enhance the activity without enantioselectivity reduction. Syn94, from Synechocystis sp. PCC 6803 was identified, and it demonstrated good enantioselectivity (>99.9 % ee) for synthesizing (S)-N-Boc-3-pyrrolidinol ((S)-Boc-PL), a key intermediate of darifenacin, but with low catalytic efficiency. To enhance its activity, three regions for loop-loop interactions near the active pocket were identified for mutagenesis. After the iteration of two effective sites, the best mutant, S89T/P282R, with a 94.5-fold enhancement in catalytic efficiency, was successfully obtained. By establishing a co-expression system of S89T/P282R and glucose dehydrogenase (GDH), up to 1.0 M (180.2 g/L) substrate could be completely reduced to (S)-Boc-PL within 7 h, with 617 g/L/d space-time-yield. Molecular dynamics simulations revealed that the enhanced activity was related to the stabilization of two loops, D41-V58 and W87-T112.
