Functional Characterization and Structural Basis of an Efficient Di-C-glycosyltransferase from Glycyrrhiza glabra

Abstract

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

Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides

“…Generally, most of the known GTs, including OGTs and CGTs, exhibit the highest catalytic activity at pH 7.0-9.0 and lower catalytic activity at pH >10.0 15,[23][24][25][26][27][28][29][30][31][32] . However, the maximum catalytic activity of AbCGT was observed at pH 11.0 (Supplementary Fig.…”

“…Investigation of the substrate promiscuity of AbCGT. Plant CGTs usually recognize only substrates with the basic core structure of acyl phloroglucinol, which possesses three hydroxyl groups and one acyl group 15,[23][24][25][26]29,31,32 . Thus, to explore the effects of the number and position of phenolic hydroxyl groups on the catalytic activity of AbCGT, an acceptor library containing acyl phenols (1-3), acyl resorcinols (4-9), acyl phloroglucinols (10-19 and 23), phloroglucinol derivatives (20)(21)(22) without basic acyl groups, and coumarins (24)(25)(26) was used for the reactions (Fig.…”

“…Acyl groups, as electron-donating and potential positioning groups, are necessary for the substrates of known plant CGTs 15,[23][24][25][26][27][28][29][30][31][32] . Among the substrates of acyl resorcinols, which should be both C-and O-glycosylated according to the above catalytic characteristics of AbCGT, there was an exception that 3,5-dihydroxyacetophenone (4) was only O-glycosylated (Supplementary Fig.…”

“…From 2009 to 2020, about ten plant CGTs were identified 15,[23][24][25][26][27][28][29][30][31] . Recently, a highly promiscuous CGT (GgCGT) with C-, O-, N-, and S-glycosylation activity was identified from Glycyrrhiza glabra 32 . GgCGT could efficiently catalyze C-glycosylation of substrates containing a flopropione unit.…”

“…GgCGT could efficiently catalyze C-glycosylation of substrates containing a flopropione unit. However, these discovered CGTs function primarily on C-glycosylating flavonoids [23][24][25][26][27][28][29][30] and other phenols with acyl groups 15,31,32 . In our previous work, although the substrate scopes of the mutated variants of a Mangifera indica CGT were expanded, the structural diversity of substrates is still limited, for example, an acyl substituent of phenolic acceptors is necessary for C-glycosylation 31 .…”