Poor water solubility limits the application of genistein in food and pharmaceutical fields. Glycosylation can greatly improve the water solubility of genistein by attaching long chain sugars. Here, glycosylation of sophoricoside (genistein monoglucoside) by cyclodextrin glycosyltransferase from Paenibacillus macerans ( Pm CGTase) was investigated. Based on molecular docking and hydrolytic activity analysis, saturation mutagenesis was conducted at residues Y195 and S77 of Pm CGTase. Variants S77N, Y195S, Y195I, S77N/Y195S, and S77N/Y195I showed enhanced product specificity toward long-chain glycosylated sophoricosides (LCGS). In particular, S77N/Y195I displayed 119% increased LCGS product specificity and 20.6% higher sophoricoside conversion than the wild type. Increased comprehensive activities of Y195S and Y195I contributed to enhanced conversion, and decreased hydrolytic activity of S77N promoted LCGS accumulation. Kinetics results revealed improved catalytic efficiency and substrate affinity of variants toward both the glycosyl donor and acceptor. Furthermore, MD simulation elucidates that the decreased substrate binding energy of variants could lead to improved kinetics, and increased hydrogen binding between residues at -6/-7 subsites and the glycosyl donor is primarily responsible for enhanced LCGS specificity. An “Increased Income-Reduced Expenditure” mechanism was proposed, explaining that the enhanced LCGS product specificity of Pm CGTase is closely related to the increased hydrogen bond interactions at -6/-7 subsites and decreased hydrolytic activity.
Jin Wang,Binhao Wang,Jieyu Zhou,Jinjun Dong,Ye Ni,Ruizhi Han
The regioselective glycosylation and product specificity of hydroxyflavonoid compounds profoundly influences their biological activity and stability, offering significant therapeutic potential. However, most cyclodextrin glucosyltransferases (CGTases) inherently lack regioselectivity and product specificity for flavone glycosylation. Herein, a CGTase fromPaenibacillus maceranswas engineered for enhanced glycosylation regioselectivity and product specificity by combining molecular docking analysis and saturation mutagenesis strategies. K232L (favoring 4′-and 6-hydroxyflavones) and K232V (favoring 7-hydroxyflavone) were identified with distinct preferences. In addition, H233Y (preferring for 4′-hydroxyflavones), H233T (preferring for 6′-hydroxyflavones), and H233K (preferring for 7′-hydroxyflavones) also demonstrated distinct regioselectivity. These variants further exhibited enhanced hydrolytic activity, enabling the efficient production of short sugar-chain glycosides. Molecular dynamics (MDs) simulations revealed that the variants adopted optimized catalytic conformations with increased loop region flexibility near the binding pocket, enhancing substrate accessibility. These findings underscore the pivotal roles of K232 and H233 in broadening the substrate scope of CGTase and offer valuable guidance for enzyme engineering targeting regioselective glycosylation.
Binhao Wang,Zhaoyue Fan,Jie Zhang,Ulrich Schwaneberg,Ye Ni,Ruizhi Han
Cyclodextrin glycosyltransferase (CGTase) is widely used in stevioside glycosylation, yet achieving precise regioselectivity remains a persistent challenge. Here, 200 ns molecular dynamics (MD) simulations identified residue Y260 in Paenibacillus macerans CGTase as a conformation-sensitive determinant for rebaudioside A (RebA) glycosylation. The Y260N variant achieved exclusive regioselectivity, producing only the C13-glycosylated products of RebA, although with a ∼30% reduction in conversion compared with wild-type. This high regioselectivity extended to other steviol glycosides─Reb C, D, and M─each yielding a single regioisomer. Further MD simulations indicated that conformational reorganization restricts access to O21 and stabilizes Glu258 toward O14, thereby enforcing C13-specific glycosylation. This work elucidates a mechanism for regioselective regulation in CGTase and provides a strategy for engineering other glycosylation enzymes.
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