甜菊糖苷是一类理想的天然甜味物质,其中莱鲍迪苷D(rebaudioside D,Reb D)因甜度高、苦味低而受到广泛关注。当前主要通过酶法以Reb A为底物,在YojK、UGTSL2、PgUGT等糖基转移酶催化下实现从Reb A到Reb D的酶法转化,然而仍缺乏对此类酶的系统性研究。该研究结合生物信息学分析与分子模拟方法,系统探讨了酶法转化所需糖基转移酶的序列与结构特征,并进一步解析酶学性能提升的分子机制。结果发现,活性中心区域结构保守性高于整体蛋白,且疏水性环状侧链残基高度保守。此外,催化效率的提升并不依赖于稳定性或氢键数量的增加,更与催化原子间距离和底物之间协同作用密切相关。能量和氢键动态网络分析发现,酶与底物Reb A的结合主要由疏水相互作用主导,两侧糖基的氢键作用显著强于苷元部分,且突变进一步增强了糖基侧的氢键作用。基于上述结果,该研究提出了底物结合机制模型,为Reb A到Reb D高效酶法转化提供了理论基础。
Steviol glycosides are ideal natural sweeteners, among which rebaudioside D (Reb D) is widely favored due to its high sweetness and low bitterness.Currently, it is mainly synthesized enzymatically from rebaudioside A (Reb A) by glycosyltransferases such as YojK, UGTSL2, and PgUGT.However, systematic studies on these enzymes remain limited.In this study, bioinformatics analysis combined with molecular simulations was used to comprehensively investigate the sequence and structural features of glycosyltransferases involved in Reb D biosynthesis, and to elucidate the molecular mechanisms underlying improved enzymatic performance.Results revealed that the active site regions exhibited higher structural conservation than the overall proteins, with highly conserved hydrophobic cyclic side-chain residues.Moreover, improved catalytic efficiency was not dependent on increased stability or hydrogen bond quantity, but was more closely related to the distance between catalytic atoms and the synergistic positioning of substrates.Energy and dynamic hydrogen bond network analyses indicated that binding between enzymes and Reb A was mainly driven by hydrophobic interactions.The hydrogen bonding interactions at both glycosyl ends were significantly stronger than those at the aglycone moiety, and the mutations further enhanced these hydrogen bond interactions.Based on these findings, a substrate binding mechanism model was proposed, providing a theoretical basis for the efficient enzymatic conversion of Reb D.
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