Functional characterization, structural basis, and regio-selectivity control of a promiscuous flavonoid 7,4'-di--glycosyltransferase from var. .

A highly efficient and promiscuous 7,4'-di--glycosyltransferase ZjOGT3 was discovered from the medicinal plant var. . ZjOGT3 could sequentially catalyse 4'- and 7--glycosylation of flavones to produce 7,4'-di--glycosides with obvious regio-selectivity. For 7,4'-dihydroxyl flavanones and 3--glycosylated 7,4'-dihydroxyl flavones, ZjOGT3 selectively catalyses 7--glycosylation. The crystal structure of ZjOGT3 was solved. Structural analysis, DFT calculations, MD simulations, and site-directed mutagenesis reveal that the regio-selectivity is mainly controlled by the enzyme microenvironment for 7,4'-dihydroxyl flavones and 3--glycosylated 7,4'-dihydroxyl flavones. For 7,4'-dihydroxyl flavanones, the selectivity is mainly controlled by intrinsic reactivity. ZjOGT3 is the first plant flavonoid 7,4'-di--glycosyltransferase with a crystal structure. This work could help understand the catalytic mechanisms of multi-site glycosyltransferases and provides an efficient approach to synthesise -glycosides with medicinal potential.