Identification and functional characterization of a new flavonoid glycosyltransferase from Rheum palmatum

Objective

To characterize a glycosyltransferase (RpUGT1) from Rheum palmatum and investigate its specificity toward flavonoid compounds.

Methods

The RpUGT1 was expressed in Escherichia coli and screened for catalytic activity against a range of flavonoid substrates using a high-throughput HPLC assay method. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) were used to determine the structure of the product. Homology modeling, molecular docking analyses and site-directed mutagenesis studies were conducted to identify key residues responsible for its function.

Results

The recombinant RpUGT1 protein exhibited catalytic activity towards various flavonoids. Notably, RpUGT1 catalyzed the glycosylation of isorhamnetin to form 3-O-glucoside and kaempferol to form 7-O-glucoside, utilizing uridine diphosphate (UDP) glucose as the sugar donor. The homology modeling and molecular docking analyses identified key residues responsible for its activity. Subsequent site-directed mutagenesis studies highlighted the crucial role of K307 in catalysis.

Conclusion

These discoveries offer valuable perspectives on the role of the UGT family and establish a groundwork for forthcoming research on the synthesis of flavonoids in plants.