Functional Characterization and Protein Engineering of a Glycosyltransferase GcCGT to Produce Flavone 6,8-Di-<i>C</i>- and 6-<i>C</i>-4′-<i>O</i>-Glycosides
Yang‐Oujie Bao, Meng Zhang, Haoran Li, Zilong Wang, Jiajing Zhou, Yang Yi, Fudong Li, Lei Ye, Hongye Li, Hongwei Jin, Chao He, Min Ye
Abstract
Herein, we discovered an efficient flavone 6- C -glycosyltransferase GcCGT from the medicinal plant Gentiana crassicaulis . GcCGT could catalyze consecutive two-step 6- C /4′- O -glycosylation of flavonoids. Homology modeling and site-directed mutagenesis yielded mutant F387K, which could catalyze the unprecedented 6- C -glycosylation of flavone 8- C -glycosides to produce 6,8-di- C -glycosides. To elucidate the catalytic mechanisms, the crystal structures of GcCGT-apo (2.10 Å) and GcCGT/UDP (2.40 Å) were resolved. Structural analysis and molecular dynamics simulations indicated that the lack of π–π stacking interaction for F387 changed the protein conformation and expanded the entrance of the substrate binding pocket. This work provided an efficient method to synthesize flavone 6,8-di- C - and 6- C -4′- O -glycosides.