Molecular mechanism of the pH-dependent dual catalytic activity of MaUGT84R1 from Morus alba.

Abstract

Glycosylation is a crucial modification in plant metabolism and is primarily catalyzed by UDP-glycosyltransferases (UGTs). Among these, the UGT84 subfamily is known to catalyze not only O-glycosylation of hydroxyl groups but also glucose esterification of carboxylic acids, but the underlying mechanism remains unclear. In this study, we identified the MaUGT84R1 gene from Morus alba and heterologously expressed it in both Escherichia coli and Nicotiana benthamiana. In vitro enzymatic assays confirmed its dual catalytic activity. Structural comparisons combined with phylogenetic analysis revealed, for the first time, a unique His-Asn-Ser catalytic triad within the UGT84 subfamily. Molecular docking elucidated the sugar acceptor binding pocket of MaUGT84R1, and site-directed mutagenesis validated the functional roles of key residues, including Phe17, His22, Phe87, Tyr90, Asn122, Phe199, and Val279. On the basis of these findings, we propose a molecular mechanism for the pH-dependent dual catalytic function of MaUGT84R1. In addition, we determined the cytosolic localization of MaUGT84R1 in mulberry protoplasts. Collectively, our study provides novel mechanistic and evolutionary insights into the dual catalytic activity of the UGT84 subfamily, and lays a foundation for future protein engineering efforts.