Molecular characterization and structural basis of a promiscuous glycosyltransferase for <i>β</i>‐(1,6) oligoglucoside chain glycosides biosynthesis

Sugar building blocks are crucial for the chemical diversity and biological activity of secondary metabolites. UDP-dependent glycosyltransferases (UGTs) play a pivotal role in the biosynthesis of glycosides in plants by catalysing the attachment of sugar moieties to various bioactive natural products. However, the biosynthesis of oligosaccharide-chain glycosides is often limited by the narrow substrate specificity of UGTs. In this study, we identify a regio-specific β-(1,6) glycosyltransferase, UGT94BY1, from Platycodon grandiflorum. UGT94BY1 exhibits broad substrate promiscuity and can transfer up to three sugar moieties to the C6-OH position of the glucosyl group in various triterpenoids and phenolic glycosides, thereby forming β-(1,6) oligoglucoside chains. To elucidate the mechanism underlying its substrate selectivity, we determined the crystal structure of the UGT94BY1 complex with UDP at a resolution of 2.0 Å. Molecular simulations revealed that a critical structural motif, comprising residues N84-M91, S141-L155 and R179-E186, plays a key role in recognizing sugar acceptors and facilitating chain elongation. Our study unveils a powerful glycosyltransferase for β-(1,6) oligoglucoside chain biosynthesis and highlights key regions involved in substrate recognition and sugar chain extension, providing valuable insights for designing UGTs with customized substrate specificities for biotechnological applications.