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

• Published in: Angewandte Chemie 2022-02, Vol.134 (8), p.n/a
• Main Authors: Zhang, Meng, Yi, Yang, Gao, Bai‐Han, Su, Hui‐Fei, Bao, Yang‐Oujie, Shi, Xiao‐Meng, Wang, Hai‐Dong, Li, Fu‐Dong, Ye, Min, Qiao, Xue
• Format: Article
• Language: English
• Subjects: Astragalus membranaceus; Biocatalysts; Catalytic mechanism; Chemistry; Conserved residue; Deuterium; Glycosylation; Glycosyltransferase; Herbal medicine; Hydrogen-deuterium exchange; Mass spectrometry; Mass spectroscopy; Medicinal plants; Molecular dynamics; Mutants; Protein engineering; Proteins; Regioselectivity; Residues; Saponins
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.202113587

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. We characterized the first plant cycloartane glycosyltransferase AmGT8 from A. membranaceus. Its mutants A394F, A394D, and T131V were discovered using semi‐rational design, which showed specific 6‐O, 3‐O, and 2′‐O glycosylation activities, respectively. This study uncovered a conserved residue critical for the regiospecificity of plant glycosyltransferases.