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.

中文翻译：

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单氢键控制家族 13 糖苷水解酶中转糖基化与水解的选择性

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) 的选择性的分子决定因素仍未完全解开。在这里，我们通过实验表明，一个活性位点残基的突变可以在两个高度同源的 GH 中切换水解和转糖基化之间的酶活性。进一步的 QM/MM 模拟表明，突变通过与亲核试剂 Asp 的单个 H 键的存在/不存在来调节 T 与 H 反应势垒。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，它取代了糖受体，导致转糖基化的亲和力降低和显着的空间排斥。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。