Techniques for synthesis of bespoke oligosaccharides currently lag behind those for other biopolymers such as polypeptides and polynucleotides, in part because of the paucity of satisfactory enzymatic tools to perform the synthetic reactions. One promising avenue of development for this problem is glycoside hydrolase enzymes with mutated nucleophile residues (called glycosynthases), which retain some elements of their native specificity and work with cheaply available substrates. However, the mechanistic underpinnings of this class of enzymes are not yet well-understood, and what few atomistic studies have been conducted have found different reaction pathways. In this paper, we describe the first unbiased computational study of the mechanism of a GH29 glycosynthase enzyme, Thermotoga maritima α-L-fucosidase (TmAfc) D224G. We find a single-step endothermic reaction step with an oxocarbenium-like transition state, demonstrating how stabilization of this transition state structure (which is common to many retaining glycoside hydrolases) can be repurposed in mutant enzymes to perform synthesis instead of hydrolysis. Our results are consistent with previous experimental observations and help both to clarify the mechanism of the existing single-mutant and to provide directions for further engineering of this and other glycosynthases.

中文翻译：

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通过突变的GH29岩藻糖苷酶 合成寡糖的机理†

定制寡糖的合成技术目前落后于其他生物聚合物（例如多肽和多核苷酸）的技术，部分原因是缺乏令人满意的酶工具来进行合成反应。解决该问题的一种有希望的途径是具有突变的亲核残基的糖苷水解酶（称为糖合酶），该酶保留其天然特异性的某些元素并与廉价的底物一起工作。然而，这类酶的机理基础尚不为人所理解，并且进行了很少的原子学研究发现了不同的反应途径。在本文中，我们描述了GH29糖苷酶的机制的第一偏见的计算研究喜温海洋α-大号-岩藻糖苷酶（Tm Afc）D224G。我们发现了一个具有氧碳鎓类过渡态的一步吸热反应步骤，证明了这种过渡态结构的稳定性（这是许多保留糖苷水解酶所共有的）如何可以用于突变酶中以进行合成而不是水解。我们的结果与以前的实验观察结果一致，并有助于阐明现有单突变体的机制，并为进一步改造该和其他糖合酶提供指导。