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

Summary 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.