Genome-Wide Characterization of the Glycosyltransferase Family in Morus alba L. and Functional Characterization of the Flavonoid-Specific MaUGT89AS1

Abstract

Mulberry is an important industrial crop and medicinal plant with a history of thousands of years. Glycosylation catalysed by glycosyltransferase (GT) is one of the most important modification reactions necessary to maintain metabolic homeostasis in plant cellular processes and is often involved in the biosynthesis of secondary metabolites. Mulberry contains a large number of active glycosylated products, especially flavonoids and stilbenes, while their biosynthesis has not been fully elucidated. In this study, a total of 121 GT encoding genes were identified in the genome of Morus alba L., and their gene structures, chromosomal locations, and expression levels were analysed. Phylogenetic analysis suggested that MaUGT89AS1 might recognize the 7-OH site of flavonoid substrates. In vitro enzymatic activity analysis showed that MaUGT89AS1 was able to glycosylate kaempferol at both 7-OH and 3-OH to form kaempferol-7-O-β-D-glucopyranoside and kaempferol-3-O-β-D-glucopyranoside. In addition, it had glycosylation activity towards 7-OH of other flavonoids such as quercetin. The optimum reaction temperature and pH of MaUGT89AS1 were 40°C and 8.0, respectively. Molecular docking elucidated the binding conformations and interactions for MaUGT89AS1 to recognize different glycosylation sites. Site-directed mutagenesis proved the essential role of His16 and Asp119 catalytic dichotomies in glycosylation reaction. This work will provide molecular resources for heterologous synthesis of flavonoid glycoside compounds with important medicinal activities.