Functional Characterization and Protein Engineering of a Triterpene 3‐/6‐/2′‐O‐Glycosyltransferase Reveal a Conserved Residue Critical for the Regiospecificity

Abstract Engineering the function of triterpene glucosyltransferases (GTs) is challenging due to the large size of the sugar acceptors. In this work, we identified a multifunctional glycosyltransferase AmGT8 catalyzing triterpene 3‐/6‐/2′‐ O ‐glycosylation from the medicinal plant Astragalus membranaceus . To engineer its regiospecificity, a small mutant library was built based on semi‐rational design. Variants A394F, A394D, and T131V were found to catalyze specific 6‐ O , 3‐ O , and 2′‐ O glycosylation, respectively. The origin of regioselectivity of AmGT8 and its A394F variant was studied by molecular dynamics and hydrogen deuterium exchange mass spectrometry. Residue 394 is highly conserved as A/G and is critical for the regiospecificity of the C ‐ and O ‐GTs TcCGT1 and GuGT10/14. Finally, astragalosides III and IV were synthesized by mutants A394F, T131V and P192E. This work reports biocatalysts for saponin synthesis and gives new insights into protein engineering of regioselectivity in plant GTs.