Enhancing Rebaudioside\nM Synthesis via Introducing\nSulfur-Mediated Interactions between Glycosyltransferase UGT76G1 and\nRebaudioside D

Rebaudioside\nM (Reb M), a zero-calorie sweetener with\nhigh sweetness,\nfaces production challenges due to its low yield and purity. UGT76G1,\na uridine diphosphate glucose (UDPG)-dependent glycosyltransferase,\nforms a β-1,3-glycosidic bond with rebaudioside D (Reb D) to\nproduce Reb M but with an efficiency lower than that for stevioside\n(ST). This study identified the variant UGT76G1-L200A/L379 M (No.11),\nwhich exhibited a 10-fold increase in enzymatic activity toward Reb\nD compared to wild-type UGT76G1 (WT). Coupled with mbSUS, the No.11\neffectively synthesized Reb M, achieving a 96.85% yield from 34.89\nmM Reb D in 60 min at 50 °C. Molecular dynamics revealed the\nmolecular mechanism behind this enhanced catalytic activity: the No.11,\nUGT76G1-L200A, and UGT76G1-L379 M complexes showed shorter and more\nstable interactions between Reb D-C<sub>19</sub>-Glc<sub>1</sub>-3-hydroxyl,\ncatalytic residue H20, and UDPG-C1’ compared to WT. The root-mean-square\nfluctuation (RMSF) values and binding free energy analyses further\nexplained the No.11’s superior catalytic efficiency. This study\nintroduces a novel protein engineering approach by introducing specific\namino acids to trigger nonclassical interactions, improving ligand–protein\nbinding and catalysis.