Abstract：
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.
摘要：
从基本观点和实际应用来看，了解酶特异性的机制越来越重要。由于其在体内和体外改善受体底物的功能特性方面的重要性，转糖基化引起了许多关注。环糊精葡聚糖转移酶(CGTase)是转糖基化的关键酶之一。它具有广泛的底物谱，并利用糖作为供体。然而,对CGTase的受体选择性知之甚少，这极大地阻碍了合理设计所需的转糖基化衍生物的努力。在这项研究中,我们发现来自环状芽孢杆菌的CGTase，BcCGTase,能够与多种人参皂苷形成糖基化产物。特别是,它不仅进行不同的单一，di-,甚至通过将葡萄糖部分转移到COGlc位置进行更高阶的糖基化，还可以糖基化人参皂苷的C3-OH位置。相比之下,地衣芽孢杆菌的另一种CGTase（BlCGTase）显示出相对特定的受体偏好，只有几种人参皂苷。BcCGTase和BlCGTase之间的结构比较表明，BcCGTase/BlCGTase受体结合位点内的Arg74/K81位置是人参皂苷F1催化特异性差异的原因。进一步的诱变证实了它们在受体选择中的作用。总之,我们的研究不仅证明了CGTases的受体选择性，而且还提供了对CGTases催化机理的见解，这将潜在地增加CGTase生物合成新的，合理设计的转糖基化衍生物。
