Molecular basis of ligand recognition specificity of flavone glucosyltransferases in Nemophila menziesii

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-beta-glucoside-4 '-O-beta-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4 '-O-glucosyltransferase (NmF4 ' GT) and UDP-glucose:flavone 4 '-O-glucoside 7-O-glucosyltransferase (NmF4 ' G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4 ' G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4 ' GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4 ' G7GT. The substrate specificity of NmF4 ' GT was examined by comparing its model structure with that of NmF4 ' G7GT. The structure of NmF4 ' GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).