A highly selective C-rhamnosyltransferase from Viola tricolor and insights into its mechanisms

Abstract

<i>C</i>-Glycosides are important natural products with various bioactivities. In plant biosynthetic pathways, the <i>C</i>-glycosylation step is usually catalyzed by <i>C</i>-glycosyltransferases (CGTs), and most of them prefer to accept uridine 5'-diphosphate glucose (UDP-Glc) as sugar donor. No CGTs favoring UDP-rhamnose (UDP-Rha) as sugar donor has been reported, thus far. Herein, we report the first selective <i>C</i>-rhamnosyltransferase VtCGTc from the medicinal plant <i>Viola tricolor</i>. VtCGTc could efficiently catalyze <i>C</i>-rhamnosylation of 2-hydroxynaringenin 3-<i>C</i>-glucoside, and exhibited high selectivity towards UDP-Rha. Mechanisms for the sugar donor selectivity of VtCGTc were investigated by molecular dynamics (MD) simulations and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) binding free energy calculations. Val144 played a vital role in recognizing UDP-Rha, and the V144T mutant could efficiently utilize UDP-Glc. This work provides a new and efficient approach to prepare flavonoid <i>C</i>-rhamnosides such as violanthin and iso-violanthin.