Characterization of a UGT84 Family Glycosyltransferase Provides New Insights into Substrate Binding and Reactivity of Galloylglucose Ester-Forming UGTs.

Galloylated plant specialized metabolites play important roles in plant-environment interactions and in the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs. By employing site-directed mutagenesis of candidate amino acids, enzyme assays with analogous substrates, and kinetic analysis, we elucidated key amino acid sites for PgUGT84A23 substrate binding and reactivity. The galloylglucose ester-forming UGT84s have not been shown to glycosylate genistein (an isoflavonoid) in vivo. Unexpectedly, amino acids highly conserved among UGT84s that affect specifically the binding of genistein but not gallic acid or other tested sugar acceptors were identified. This result suggests that genistein may resemble the substrate profile for the enzyme ancestor of the galloylglucose ester-forming UGTs and recruited during transition from a general to a more specialized defense function. Overall, a better understanding of the structure-function relationship of UGT84s will facilitate enzyme engineering for the production of pharmaceutically and industrially valuable glycosylated compounds.