Research and Development Center of Chinese Medicine Resources and Biotechnology, The Ministry of Education (MOE) Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
Research and Development Center of Chinese Medicine Resources and Biotechnology, The Ministry of Education (MOE) Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang 222000, China.
Bioorg Chem. 2022 Nov;128:106094. doi: 10.1016/j.bioorg.2022.106094. Epub 2022 Aug 12.
Understanding the mechanisms of enzyme specificity is increasingly important from a fundamental viewpoint and for practical applications. Transglycosylation has attracted many attentions due to its importance in improving the functional properties of acceptor substrates both in vivo and in vitro. Cyclodextrin glucanotransferase (CGTase) is one of the key enzymes in transglycosylation, it has a broad substrate spectrum and utilizes sugar as the donor. However, little is known about the acceptor selectivity of CGTase, which greatly hampers efforts toward the rational design of desirable transglycosylated derivatives. In this study, we found that the CGTase from Bacillus circulans, BcCGTase, was able to form glycosylated products with diverse ginsenosides. In particular, it not only carries out diverse mono-, di-, and even higher-order glycosylations via the transfer of glucose moieties to the COGlc positions, but also can glycosylate the C3-OH position of ginsenosides. In contrast, another CGTase from Bacillus licheniformis (BlCGTase) showed relatively specific acceptor preference with only several ginsenosides. Structural comparison between BcCGTase and BlCGTase revealed that the Arg74/K81 position within the acceptor-binding sites of BcCGTase/BlCGTase was responsible for the differences in catalytic specificity for ginsenoside F1. Further mutagenesis confirmed their roles in the acceptor selection. In conclusion, our study not only demonstrates the acceptor selectivity of CGTases, but also provides insight into the catalytic mechanism of CGTases, which will potentially increase the utility of CGTase for biosynthesis of new, rationally designed transglycosylated derivatives.
从基础理论和实际应用的角度来看，了解酶特异性的机制变得越来越重要。由于其在体内和体外改善受体底物功能性质方面的重要性，转糖苷作用引起了广泛关注。环糊精葡萄糖基转移酶（CGTase）是转糖苷作用中的关键酶之一，它具有广泛的底物谱，并利用糖作为供体。然而，对于 CGTase 的受体选择性知之甚少，这极大地阻碍了合理设计理想的转糖苷衍生物的努力。在这项研究中，我们发现来自环状芽孢杆菌的 CGTase（BcCGTase）能够与各种人参皂苷形成糖基化产物。特别是，它不仅可以通过将葡萄糖基团转移到 COGlc 位置来进行不同的单、二甚至更高阶的糖基化，还可以糖基化人参皂苷的 C3-OH 位置。相比之下，另一种来自地衣芽孢杆菌的 CGTase（BlCGTase）则表现出相对特定的受体偏好，只能与几种人参皂苷作用。BcCGTase 和 BlCGTase 之间的结构比较表明，BcCGTase/BlCGTase 受体结合位点内的 Arg74/K81 位置负责催化特异性对人参皂苷 F1 的差异。进一步的突变分析证实了它们在受体选择中的作用。总之，我们的研究不仅展示了 CGTase 的受体选择性，还深入了解了 CGTase 的催化机制，这将有可能增加 CGTase 在新型合理设计的转糖苷衍生物生物合成中的应用。
