Engineering the Entrance of a Flavonoid Glycosyltransferase Promotes the Glycosylation of Etoposide Aglycone.

Enzyme entrances, which function as the first molecular filters, influence substrate selectivity and enzymatic activity. Because of low binding affinities, engineering enzyme entrances that recognize non-natural substrates is a major challenge for artificial biocatalyst design. Here, the entrance of flavonoid glycosyltransferase UGT78D2 was engineered to promote the recognition of the aglycone of etoposide, a chemotherapeutic agent. We found that Q258, S446, R444, and R450, the key residues surrounding the substrate entrance, specifically guide the flux of etoposide aglycone, which has a high steric hindrance, into the active site; this activity was inferred to be determined by the entrance size and hydrophobic and electrostatic interactions. Engineering the coordination of Q258 and S446 to increase the entrance size and hydrophobic interaction between UGT78D2 and etoposide aglycone increased the affinity by 10.10-fold and the conversion by 10%. The entrance-engineering strategy applied in this study can improve the design of artificial biocatalysts.

The Advances and Challenges in Enzymatic C-glycosylation of Flavonoids in Plants.

Flavonoid glycosides play determinant roles in plants and have considerable potential for applications in medicine and biotechnology. Glycosyltransferases transfer a sugar moiety from uridine diphosphateactivated sugar molecules to an acceptor flavonoid via C-O and C-C linkages. Compared with O-glycosyl flavonoids, C-glycosyl flavonoids are more stable, resistant to glycosidase or acid hydrolysis, exhibit better pharmacological properties, and have received more attention. In this study, we discuss the mining of C-glycosyl flavones and the corresponding C-glycosyltransferases and evaluate the differences in structure and catalytic mechanisms between C-glycosyltransferase and O-glycosyltransferase. We conclude that promiscuity and specificity are key determinants for general flavonoid C-glycosyltransferase engineering and summarize the C-glycosyltransferase engineering strategy. A thorough understanding of the properties, catalytic mechanisms, and engineering of C-glycosyltransferases will be critical for future biotechnological applications in areas such as the production of desired C-glycosyl flavonoids for nutritional or medicinal use.