Cyclodextrin glycosyltransferase (CGTase) catalyzes intermolecular transglycosylation through either disproportionation or cyclization-coupling pathway. Kinetics analysis reveals that the hesperidin glycosylation process catalyzed by a CGTase variant (M1) is primarily accomplished through the disproportionation pathway. The cyclization-coupling pathway exhibits a lower reaction rate and competitively consumes glycosyl donor and yield byproducts that impair disproportionation. Under the guidance of reaction kinetics, mutagenesis was targeted at residues in the −3, +1, and +2 subsites, known to control the selectivity between disproportionation and cyclization. A quadruple variant was identified with 2.9 times hesperidin glycosylation activity compared to M1, and 20.3 times compared to the wild-type. Kinetic analysis reveals a fourfold improvement of k_cat/K_mA for disproportionation and an 85.5% reduction in k_cat/K_m for cyclization after mutagenesis. Binding free energy analysis further confirms that the mutagenesis favors the binding of hesperidin, and destabilizes the binding of cyclodextrin.

中文翻译：

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具有增强分子间转糖基活性的环糊精糖基转移酶的动力学指导工程


环糊精糖基转移酶 (CGTase) 通过歧化或环化偶联途径催化分子间转糖基化。动力学分析表明，CGTase 变体 (M1) 催化的橙皮苷糖基化过程主要通过歧化途径完成。环化偶联途径表现出较低的反应速率并竞争性消耗糖基供体并产生损害歧化的副产物。在反应动力学的指导下，诱变针对-3、+1和+2亚位点中的残基，已知这些残基控制歧化和环化之间的选择性。鉴定出四重变体，其橙皮苷糖基化活性是 M1 的 2.9 倍，是野生型的 20.3 倍。动力学分析表明，歧化反应的 k _cat /K _mA 提高了四倍，并且 k _cat /K _m 降低了 85.5%诱变后环化。结合自由能分析进一步证实诱变有利于橙皮苷的结合，并破坏环糊精的结合的稳定性。