Kinetics guided engineering of cyclodextrin glycosyltransferase with enhanced intermolecular transglycosylation activity

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.