Conformation-Sensitive Active-Site Residue Tyrosine 260 in <i>Paenibacillus macerans</i> Cyclodextrin Glycosyltransferase Governs C13-Regioselective Glycosylation of Rebaudioside A

Cyclodextrin glycosyltransferase (CGTase) is widely used in stevioside glycosylation, yet achieving precise regioselectivity remains a persistent challenge. Here, 200 ns molecular dynamics (MD) simulations identified residue Y260 in <i>Paenibacillus macerans</i> CGTase as a conformation-sensitive determinant for rebaudioside A (RebA) glycosylation. The Y260N variant achieved exclusive regioselectivity, producing only the C13-glycosylated products of RebA, although with a ∼30% reduction in conversion compared with wild-type. This high regioselectivity extended to other steviol glycosides─Reb C, D, and M─each yielding a single regioisomer. Further MD simulations indicated that conformational reorganization restricts access to O21 and stabilizes Glu258 toward O14, thereby enforcing C13-specific glycosylation. This work elucidates a mechanism for regioselective regulation in CGTase and provides a strategy for engineering other glycosylation enzymes.