Cloning, Expression, and Activity and Structure Characterization of an O-Glycosyltransferase, ZmUGT76B1 from Maize...

Flavonoids, secondary phytometabolites, play many roles in human health and plant resistance, growth, and development. They are commonly glycosylated in the last step of their biosynthesis. In this study, a gene encoding an efficient flavonoid glycosyltransferase, ZmUGT76B1, was cloned from maize and expressed. The enzyme activity and function were characterized. ZmUGT76B1 catalyzed selective glycosylation at 3-, 7-, 3'-, and 4'-hydroxyl sites of flavonoids to form mono-O-glycoside and di-O-glycoside products. Moreover, ZmUGT76B1 had a broad substrate preference and strict sugar donor selectivity. Guided by simulated molecular docking and simulated mutagenesis, three ZmUGT76B1 mutants, Q338P, E361P, and Q338P/E361P, were constructed. The mutation of either of the two sites substantially reduced the enzyme activity, whereas the double mutation eliminated the activity. The determination of enzyme activity and prediction of the active site of this enzyme have expanded our knowledge of maize uridine diphosphate-dependent glycosyltransferases, providing an important glycosyltransferase for metabolic engineering of mono- and dioxygenated glucosyl flavonoids.