Exploring the catalytic function and active sites of a novel C-glycosyltransferase from Anemarrhena asphodeloides

Anemarrhena asphodeloides is an immensely popular medicinal herb in China,which contains an abundant of mangiferin.As an important bioactive xanthone C-glycoside,mangiferin possesses a variety of pharmacological activities and is derived from the cyclization reaction of a benzophenone C-glycoside(maclurin).Biosyntheti-cally,C-glycosyltransferases are critical for the formation of benzophenone C-glycosides.However,the benzo-phenone C-glycosyltransferases from Anemarrhena asphodeloides have not been discovered.Herein,a promiscuous C-glycosyltransferase(AaCGT)was identified from Anemarrhena asphodeloides.It was able to catalyze efficiently mono-C-glycosylation of benzophenone,together with di-C-glycosylation of dihydrochalcone.It also exhibited the weak O-glycosylation or potent S-glycosylation capacities toward 12 other types of flavonoid scaffolds and a simple aromatic compound with–SH group.Homology modeling and mutagenesis experiments revealed that the glycosylation reaction of AaCGT was initiated by the conserved residue H23 as the catalytic base.Three critical residues H356,W359 and D380 were involved in the recognition of sugar donor through hydrogen-bonding interactions.In particular,the double mutant of F94W/L378M led to an unexpected enzy-matic conversion of mono-C-to di-C-glycosylation.This study highlights the important value of AaCGT as a potential biocatalyst for efficiently synthesizing high-value C-glycosides.

A multi-enzymatic cascade reaction for the synthesis of bioactive C-oligosaccharides

C-Oligosaccharides are rare in nature and possess diverse bioactivities.However,their chemical synthesis faces many challenges.In this work,enzymatic introduction of C-linked sugar chains to target aglycones was successfully achieved by multi-enzymatic cascade reactions.A C-glycosyltransferase from Aloe barbadensis was employed to introduce the first C-linked glucose and then a cyclomaltodextrin glucanotransferase from Bacillus licheniformis was used to extend the sugar chain.A total of twenty C-oligosaccharides with 2-6 sugars were synthesized from scale-up reactions and exhibited good water solubility and sodium-dependent glucose transporter 2(SGLT2)inhibitory activity.Furthermore,a glucoamylase was used to control the length of the sugar chain and the C-maltosides were efficiently synthesized.These findings not only expanded the structural diversity of C-oligosaccharides,but also provided a strategy for the modification of C-glycoside drugs to improve the druggability.