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.
Subject(s)
Glycosyltransferases/metabolism , Protein Engineering , Saponins/biosynthesis , Triterpenes/metabolism , Astragalus propinquus/enzymology , Biocatalysis , Glycosyltransferases/chemistry , Protein Conformation , Saponins/chemistry , Stereoisomerism , Triterpenes/chemistryABSTRACT
Huang-Qi (Astragali Radix) is an herbal tonic widely used in China and many other countries. It is derived from the roots of Astragalus membranaceus and A. membranaceus var. mongholicus and shows potent cardiovascular protective effects. In this article, we comprehensively reviewed 189 small molecules isolated from the two Astragalus species and discussed the interspecies chemical differences. Moreover, we summarized the pharmacological activities and mechanisms of action of Huang-Qi and its major bioactive compounds for the treatment of cardiovascular diseases. This review covers 171 references published between February 1983 and March 2020.
Subject(s)
Astragalus Plant , Astragalus propinquus , China , Humans , Plant RootsABSTRACT
A highly efficient di-C-glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra. GgCGT catalyzes a two-step di-C-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-C-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-C-glycosylation. GgCGT is the first di-C-glycosyltransferase with a crystal structure, and the first C-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C-glycosides with medicinal potential.