Enhancing Rebaudioside M Synthesis via Introducing Sulfur-Mediated Interactions between Glycosyltransferase UGT76G1 and Rebaudioside D

Rebaudioside M (Reb M), a zero-calorie sweetener with high sweetness, faces production challenges due to its low yield and purity. UGT76G1, a uridine diphosphate glucose (UDPG)-dependent glycosyltransferase, forms a β-1,3-glycosidic bond with rebaudioside D (Reb D) to produce Reb M but with an efficiency lower than that for stevioside (ST). This study identified the variant UGT76G1-L200A/L379 M (No.11), which exhibited a 10-fold increase in enzymatic activity toward Reb D compared to wild-type UGT76G1 (WT). Coupled with mbSUS, the No.11 effectively synthesized Reb M, achieving a 96.85% yield from 34.89 mM Reb D in 60 min at 50 °C. Molecular dynamics revealed the molecular mechanism behind this enhanced catalytic activity: the No.11, UGT76G1-L200A, and UGT76G1-L379 M complexes showed shorter and more stable interactions between Reb D-C₁₉-Glc₁-3-hydroxyl, catalytic residue H20, and UDPG-C1' compared to WT. The root-mean-square fluctuation (RMSF) values and binding free energy analyses further explained the No.11's superior catalytic efficiency. This study introduces a novel protein engineering approach by introducing specific amino acids to trigger nonclassical interactions, improving ligand-protein binding and catalysis.