A Single Hydrogen Bond Controls the Selectivity of Transglycosylation vs Hydrolysis in Family 13 Glycoside Hydrolases
A Single Hydrogen Bond Controls the Selectivity of Transglycosylation vs Hydrolysis in Family 13 Glycoside Hydrolases
复制标题
单个氢键控制家族 13 糖苷水解酶中转糖基化与水解的选择性
DOI：
10.1021/acs.jpclett.2c01136
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发表时间：
2022-06-23
影响因子：
5.7
通讯作者：
Wu, Jing
中科院分区：
文献类型：
--
作者：
Guo, Zhiyong;Wang, Lei;Wu, Jing
通过转糖基化将糖苷水解酶（GH）从水解酶转化为合成酶是复杂碳水化合物生物合成的长期目标。然而，转糖基化（T）与水解（H）选择性的分子决定因素仍未完全阐明。在此，我们实验表明，一个活性位点残基的突变可以在两个高度同源的GH中在水解和转糖基化之间切换酶活性。进一步的QM/MM模拟表明，突变调制的T与H反应障碍，通过存在/不存在的一个单一的H-键与亲核试剂天冬氨酸。这种氢键通过双重作用控制产物选择性：一方面，它促进糖基-酶中间体的断裂。另一方面，它取代糖受体，导致亲和力降低和转糖基化的显著空间排斥。这些发现扩展了我们对调节GH中T/H平衡的分子机制的理解。
Converting glycoside hydrolases (GHs) from hydrolytic to synthetic enzymes via transglycosylation is a long-standing goal for the biosynthesis of complex carbohydrates. However, the molecular determinants for the selectivity of transglycosylation (T) vs hydrolysis (H) are still not fully unraveled. Herein, we show experimentally that mutation of one active site residue can switch the enzyme activity between hydrolysis and transglycosylation in two highly homologous GHs. Further QM/MM simulations reveal that the mutation modulates the T vs H reaction barriers via the presence/absence of a single H-bond with the nucleophile Asp. Such a H-bond controls the product selectivity via a dual effect: on one hand, it facilitates the breaking of the glycosyl-enzyme intermediate. On the other, it displaces the sugar acceptor, resulting in a reduced affinity and significant steric repulsion for transglycosylation. These findings expand our understanding of the molecular mechanisms that modulate the T/H balance in GHs.