Functional Characterization and Structural Basis of an Efficient Di--glycosyltransferase from .

A highly efficient di--glycosyltransferase GgCGT was discovered from the medicinal plant . GgCGT catalyzes a two-step di--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--glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono--glycosylation. GgCGT is the first di--glycosyltransferase with a crystal structure, and the first -glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize -glycosides with medicinal potential.