Functional characterization of a Stevia rebaudiana flavonoid glycosyltransferase

Stevia rebaudiana, a perennial herb, is recognized not only for its sweet steviol glycosides but also for its rich flavonoid content, which confer pharmacological properties including anti-inflammatory, antimicrobial, and anticancer activities. However, the enzymatic basis underlying flavonoid modification in S. rebaudiana remains poorly understood. In this study, we identified, cloned, and heterologously expressed a novel flavonoid glycosyltransferase gene, SrUGT72B1 in E. coli. The recombinant SrUGT72B1 catalyzed the glycosylation of multiple flavonoids using UDP-glucose as the primary sugar donor, and exhibited broad substrate promiscuity toward apigenin, luteolin, phloretin and kaempferol. In addition to UDP-glucose, SrUGT72B1 also accepted UDP-xylose and UDP-rhamnose, with UDP-glucose exhibiting the highest catalytic efficiency. Biochemical characterization revealed that the enzyme functions optimally at pH 9.0 and 50 °C. Notably, SrUGT72B1 demonstrates regioselective 5-O-glycosylation toward apigenin, a rare activity among plant glycosyltransferases. Molecular docking and molecular dynamics simulations provided structural insights into this unique regioselectivity and substrate recognition. Together, these findings establish SrUGT72B1 as a previously uncharacterized flavonoid 5-O-glycosyltransferase, expanding the functional landscape of plant UGTs and offering potential applications in the biosynthesis of value-added flavonoid glycosides.