Engineering a Medium‐chain Alcohol Dehydrogenase for Efficient Synthesis of (S)‐N−Boc‐3−pyrrolidinol by Adjusting the Conformational Dynamics of Loops

Abstract 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.