A comparative molecular dynamics approach guides the tailoring of glycosyltransferases to meet synthetic applications†

The properties of natural products can be significantly influenced by glycosylation, emphasizing the key role of glycosyltransferases (GTs) in this process. The pursuit of tailored GT catalysts to meet the demands of the glycosylation industry aligns with the principles of green chemistry. However, steering GT engineering towards the desired direction often requires substantial effort. Herein, we employ a comparative molecular dynamics approach to guide the engineering of GTs to alter their catalytic performances. Through comparing the structural flexibility and site-saturation mutagenesis of two GTs BarGT-1 with narrow substrate scope and BarGT-3 with wide substrate scope, the identified substitution K321P in C-loop 5 of BarGT-1 greatly expanded the substrate scope towards diverse pharmaceutically valuable substrates, thereby the catalytic efficiencies were remarkably improved (e.g., 52- and 244-fold for 4′- and 6-hydroxyflavone, k_cat/K_M). Further, phylogenetic analysis demonstrated that lysine was a highly conserved residue in the GTs within BarGT-1 branch, and its key roles in regulating the substrate scope were validated through site-saturation mutagenesis in two novel GTs, BsyGT (K321F) and BgoGT (K322W). Moreover, the corresponding substitutions G325R/D in C-loop 5 of BarGT-3 also served as switches to regulate the substrate profile of BarGT-3. Finally, through the utilization of BarGT-1 and K321P, we successfully regulated the synthesis of valuable liquiritigenin glycosides with minor effort. The comparative molecular dynamics approach provides insights into the structural dynamics of GTs, optimizes enzymatic processes, and facilitates precise enzyme engineering, offering a directed synthetic paradigm to produce valuable glycosides.