Site-specific α-glycosylation of hydroxyflavones and hydroxyflavanones by amylosucrase from Deinococcus geothermalis

Amylosucrase (ASase) is a unique multifunctional enzyme exhibiting transglycosylation activity. In this study, the specificity of the transglycosylation activity of ASase was evaluated using several hydroxyflavones (HFVOs) and hydroxyflavanones (HFVAs). Our results reveal that the 3-OH and 7-OH positions of the mono-HFVOs and -HFVAs are resistant to transglycosylation by Deinococcus geothermalis ASase (DGAS), whereas the 6-OH and 4'-OH positions of the mono-HFVOs and -HFVAs exhibit relatively strong transglycosylation reactivities with the glucose donors released from sucrose by DGAS. Particularly, the 6-OH position is considerably more reactive (54-fold higher k_cat) than the 4'-OH position in both HFVOs and HFVAs. Further, the transglycosylation reactions with di- and tri-HFVOs and HFVAs were also investigated and observed to exhibit similar results to those observed for the mono-HFVO and -HFVA molecules. The pH of the reaction influences the reactivity of certain hydroxyl residues, indicating that the pKa values of the hydroxyl groups may be crucial factors in the transglycosylation reactions. These observations help us understand the specificity of the transglycosylation activity of ASase and to predict the transglycosylation products of flavonoids.