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.
Herein, the catalytic promiscuity of TcCGT1, a new C‐glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regio‐specifically catalyze the 8‐C‐glycosylation of 36 flavones and other flavonoids and could also catalyze the O‐glycosylation of diverse phenolics. The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 Å resolution. Molecular docking revealed a new model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate. The spacious binding pocket explains the substrate promiscuity, and the binding pose of the substrate determines C‐ or O‐glycosylation activity. Site‐directed mutagenesis at two residues (I94E and G284K) switched C‐ to O‐glycosylation. TcCGT1 is the first plant CGT with a crystal structure and the first flavone 8‐C‐glycosyltransferase described. This provides a basis for designing efficient glycosylation biocatalysts.
Herein, the catalytic promiscuity of TcCGT1, anew C-glycosyltransferase (CGT) from the medicinal plant Trollius chinensis is explored. TcCGT1 could efficiently and regiospecifically catalyze the 8-C-glycosylation of 36 flavones and other flavonoids and could also catalyze the O-glycosylation of diverse phenolics.The crystal structure of TcCGT1 in complex with uridine diphosphate was determined at 1.85 resolution. Molecular docking revealed an ew model for the catalytic mechanism of TcCGT1, which is initiated by the spontaneous deprotonation of the substrate.T he spacious binding pocket explains the substrate promiscuity,and the binding pose of the substrate determines C-or O-glycosylation activity.S itedirected mutagenesis at two residues (I94E and G284K) switched C-to O-glycosylation. TcCGT1 is the first plant CGT with ac rystal structure and the first flavone 8-Cglycosyltransferase described. This provides abasis for designing efficient glycosylation biocatalysts.
Licorice
(Glycyrrhiza uralensis) is a popular
medicinal plant containing more than 70 flavonoid and triterpenoid
glycosides. Thus far, only a few reports are available on the glycosylation
enzymes involved in their biosynthesis. In this work, we mined the
transcriptome data of G. uralensis and discovered
43 candidate genes for O-glycosyltransferase (O-GT). Among them, 17 genes could be expressed in E. coli, and functions of the enzymes were analyzed by catalyzing
eight native substrates. As a result, we characterized 11 O-GTs, including isoflavone 7-O-GTs, flavonol
3-O-GTs, and promiscuous O-GTs catalyzing
flavones, chalcones, and triterpenoids. They could efficiently synthesize
key licorice compounds such as liquiritin, isoliquiritin, ononin,
and 3-O-β-d-glucuronosyl glycyrrhetinic
acid. The diversity of O-GTs contributes to the biosynthesis
of various glycosides in licorice. These enzymes could also be used
as biocatalytic tools to synthesize other bioactive O-glycosides.
The regiospecific glycosylation of pentacyclic triterpenoids by UGT73F17, a new glycosyltransferase from Glycyrrhiza uralensis, is highlighted. UGT73F17 exhibited strict substrate specificity toward the carboxyl group at C-30/C-29 of pentacyclic triterpenoids, and showed high promiscuity to sugar donors. UGT73F17 represents the first identified triterpenoid 30/29-O-glycosyltransferase, and could be used as an effective biocatalyst to synthesize glycosyl ester saponins.
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