Institute of Chemistry and Center for Computing in Engineering and Sciences, University of Campinas-Unicamp, Campinas 13084-862, Sao Paulo, Brazil.
Institute of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Rio de Janeiro, Brazil.
J Chem Inf Model. 2021 Apr 26;61(4):1902-1912. doi: 10.1021/acs.jcim.0c01380. Epub 2021 Mar 24.
Glycoside hydrolases (GH) cleave carbohydrate glycosidic bonds and play pivotal roles in living organisms and in many industrial processes. Unlike acid-catalyzed hydrolysis of carbohydrates in solution, which can occur either via cyclic or acyclic oxocarbenium-like transition states, it is widely accepted that GH-catalyzed hydrolysis proceeds via a general acid mechanism involving a cyclic oxocarbenium-like transition state with protonation of the glycosidic oxygen. The GH45 subfamily C inverting endoglucanase from (PcCel45A) defies the classical inverting mechanism as its crystal structure conspicuously lacks a general Asp or Glu base residue. Instead, PcCel45A has an Asn residue, a notoriously weak base in solution, as one of its catalytic residues at position 92. Moreover, unlike other inverting GHs, the relative position of the catalytic residues in PcCel45A impairs the proton abstraction from the nucleophilic water that attacks the anomeric carbon, a key step in the classical mechanism. Here, we investigate the viability of an endocyclic mechanism for PcCel45A using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, with the QM region treated with the self-consistent-charge density-functional tight-binding level of theory. In this mechanism, an acyclic oxocarbenium-like transition state is stabilized leading to the opening of the glucopyranose ring and formation of an unstable acyclic hemiacetal that can be readily decomposed into hydrolysis product. characterization of the Michaelis complex shows that PcCel45A significantly restrains the sugar ring to the C chair conformation at the -1 subsite of the substrate binding cleft, in contrast to the classical exocyclic mechanism in which ring puckering is critical. We also show that PcCel45A provides an environment where the catalytic Asn92 residue in its standard amide form participates in a cooperative hydrogen bond network resulting in its increased nucleophilicity due to an increased negative charge on the oxygen atom. Our results for PcCel45A suggest that carbohydrate hydrolysis catalyzed by GHs may take an alternative route from the classical mechanism.
糖苷水解酶（GH）能够切割碳水化合物糖苷键，在生物体和许多工业过程中都发挥着关键作用。与溶液中酸催化的碳水化合物水解不同，后者可以通过环状或非环状的氧杂碳正离子样过渡态发生，人们普遍认为 GH 催化的水解是通过涉及环状氧杂碳正离子样过渡态的广义酸机制进行的，其中糖苷氧质子化。来自（PcCel45A）的 GH45 反转内切葡聚糖酶的 C 家族亚家族违背了经典的反转机制，因为其晶体结构明显缺乏一般的 Asp 或 Glu 碱基残基。相反，PcCel45A 的催化残基之一位置 92 上有一个天冬酰胺残基，天冬酰胺残基在溶液中是一种众所周知的弱碱。此外，与其他反转 GH 不同，PcCel45A 中催化残基的相对位置会阻碍从攻击糖苷碳原子的亲核水中的质子提取，这是经典机制中的关键步骤。在这里，我们使用混合量子力学/分子力学（QM/MM）模拟来研究 PcCel45A 的内循环机制的可行性，其中 QM 区域采用自洽电荷密度泛函紧密结合理论处理。在这种机制中，稳定了非环状的氧杂碳正离子样过渡态，导致吡喃葡萄糖环打开，并形成不稳定的非环状半缩醛，该半缩醛很容易分解为水解产物。对迈克尔is 复合物的表征表明，与经典的外循环机制不同，PcCel45A 显著将糖环限制在底物结合裂缝的-1 亚位的 C 椅构象，在经典的外循环机制中，环的构象是关键的。我们还表明，PcCel45A 提供了一种环境，其中其标准酰胺形式的催化天冬酰胺 92 残基参与协同氢键网络，导致氧原子上的负电荷增加，从而增加其亲核性。我们对 PcCel45A 的研究结果表明，GH 催化的碳水化合物水解可能采用与经典机制不同的替代途径。
