RESUMO
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.
Assuntos
Glicosiltransferases/química , Glicosiltransferases/metabolismo , Glycyrrhiza/enzimologia , Clonagem Molecular , Cristalografia por Raios X , Glicosilação , Glicosiltransferases/genética , Glycyrrhiza/genética , Ligantes , Modelos Moleculares , Floretina/química , Floretina/metabolismo , Especificidade por Substrato , Transcriptoma , Uridina Difosfato Galactose/química , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato Ácido Glucurônico/química , Uridina Difosfato Ácido Glucurônico/metabolismo , Uridina Difosfato N-Acetilglicosamina/química , Uridina Difosfato N-Acetilglicosamina/metabolismo , Uridina Difosfato Xilose/química , Uridina Difosfato Xilose/metabolismoRESUMO
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-ß-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.
Assuntos
Flavonoides/metabolismo , Glicosídeos/metabolismo , Glicosiltransferases/metabolismo , Glycyrrhiza uralensis/metabolismo , Triterpenos/metabolismo , Chalconas/metabolismo , Cromatografia Líquida de Alta Pressão , Glicosilação , Ácido Glicirretínico/metabolismo , Glycyrrhiza/metabolismo , Isoflavonas/metabolismo , FilogeniaRESUMO
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 1.85â 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.