| Summary: | Cucurbitacins, a group of diverse tetracyclic triterpenes, display a variety of biological effects. Glycosylation mediated by glycosyltransferases (UGTs) plays a vital role in structural and functional diversity of natural products and influences their biological activities. In this study, GT-SM, a mutant of UGT74AC1 from <i>Siraitia grosvenorii</i>, was chosen as a potential catalyst in glycosylation of cucurbitacins, and its optimal pH, temperature, and divalent metal ions were detected. This enzyme showed high activity (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>, 120 s<sup>−1</sup> µM<sup>−1</sup>) toward cucurbitacin F 25-O-acetate (CA-F<sub>25</sub>) and only produced CA-F<sub>25</sub> 2-O-β-<span style="font-variant: small-caps;">d</span>-glucose which was isolated and confirmed by <sup>1</sup>D and <sup>2</sup>D nuclear magnetic resonance. A pathway for uridine diphosphate glucose (UDP-Glc) regeneration and cucurbitacin glycoside synthesis was constructed by combing GT-SM and sucrose synthase to cut down the costly UDP-Glc. The molar conversion of CA-F<sub>25</sub> was 80.4% in cascade reaction. Molecular docking and dynamics simulations showed that CA-F<sub>25</sub> was stabilized by hydrophobic interactions, and the C2-OH of CA-F<sub>25</sub> showed more favorable catalytic conformation than that of C3-OH, explaining the high regioselectivity toward the C2-OH rather than the ortho-C3-OH of CA-F<sub>25</sub>. This work proved the important potential application of UGT74AC1 in cucurbitacins and provided an understanding of glycosylation of cucurbitacins.
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