State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing, 100191, China.
National Science Center for Physical Sciences at Microscale, Division of Molecular & Cell Biophysics and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Angew Chem Int Ed Engl. 2022 Feb 14;61(8):e202113587. doi: 10.1002/anie.202113587. Epub 2021 Dec 29.
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.
由于糖受体体积较大，因此对三萜糖基转移酶（GTs）的功能进行工程改造具有挑战性。在这项工作中，我们从药用植物黄芪中鉴定出一种多功能糖基转移酶 AmGT8，可催化三萜 3-/6-/2'-O-糖基化。为了对其区域选择性进行工程改造，我们基于半理性设计构建了一个小型突变体文库。发现变体 A394F、A394D 和 T131V 分别催化特定的 6-O、3-O 和 2'-O 糖基化。通过分子动力学和氘氢交换质谱研究了 AmGT8 及其 A394F 变体的区域选择性起源。残基 394 高度保守为 A/G，对 C 和 O-GTs TcCGT1 和 GuGT10/14 的区域特异性至关重要。最后，通过突变体 A394F、T131V 和 P192E 合成了黄芪苷 III 和 IV。这项工作报道了用于皂苷合成的生物催化剂，并为植物 GTs 的区域选择性蛋白质工程提供了新的见解。
