Molecular dynamics guided mining and engineering of glucosyltransferase in synthesizing menthyl‐β‐glucoside

Abstract This study presents an approach combining molecular dynamics (MD) simulations and near‐attack conformation (NAC) analysis to mine and engineer UDP‐glucosyltransferases. Using the MD‐NAC strategy, we identified that Bs YjiC from Bacillus subtilis possesses menthol glycosylating activity for the first time, and it exhibited a 28% higher k cat / K M value compared to the known l‐ menthol glycosylating enzyme, Bl YjiC from Bacillus licheniformis . The strategy was further employed to modify Bs YjiC by preventing nonproductive substrate motion and stabilizing NACs through engineering key residues identified by MD. Based on this strategy, 14 positive mutants were identified out of 16 candidates, and the double mutant A76D/V108L (M2) demonstrated a 10.64‐fold increase in k cat / K M value compared to the starting point. Whole‐cell cascade reactions with sucrose synthase yield 160 mM glucoside after catalyzing for 96 h, demonstrating its application merit. This work underscores MD‐driven enzyme engineering as a promising strategy to overcome limitations of static structure‐based approaches.