Functional Characterization and Structural Basis of an Efficient Di-<i>C</i>-glycosyltransferase from <i>Glycyrrhiza glabra</i>

Abstract

A highly efficient di-<i>C</i>-glycosyltransferase GgCGT was discovered from the medicinal plant <i>Glycyrrhiza glabra</i>. GgCGT catalyzes a two-step di-<i>C</i>-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-<i>C</i>-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-<i>C</i>-glycosylation. GgCGT is the first di-<i>C</i>-glycosyltransferase with a crystal structure, and the first <i>C</i>-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize <i>C</i>-glycosides with medicinal potential.