College of Chemistry, MOE Key Laboratory of Green Chemistry and Technology, Sichuan University, Chengdu, Sichuan, 610064, P. R. China.
State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, Sichuan, 610041, P. R. China.
Phys Chem Chem Phys. 2022 May 18;24(19):11919-11930. doi: 10.1039/d2cp00593j.
Carbohydrate degradation catalyzed by glucoside hydrolases (GHs) is a major mechanism in biomass conversion. GH family 9 endoglucanase (Cel9G) from , a typical multimodular enzyme, contains a catalytic domain closely linked to a family 3c carbohydrate-binding module (CBM3c). Unlike the conventional behavior proposed for other carbohydrate-binding modules, CBM3c has a direct impact on catalytic activity. In this work, extensive molecular dynamics (MD) simulations were employed to clarify the functional role of CBM3c. Furthermore, the detailed catalytic mechanism of Cel9G was investigated at the atomistic level using the combined quantum mechanical and molecular mechanical (QM/MM) method. Based on these simulations, owing to the rigidity of the peptide linker, CBM3c may affect the enzymatic activity direct interactions with alpha helix 4 of GH9, especially with the K123 and H125 residues. In addition, using cellohexaose as a substrate, the QM/MM MD simulations confirmed that this enzyme can cleave the β-1,4-glycosidic linkage an inverting mechanism. An oxocarbenium ion-like transition state was located with a barrier height of 19.6 kcal mol. Furthermore, the G(-1) pyranose unit preferentially adopted a distorted S/H conformer in the enzyme-substrate complex. For the cleavage of the glycosidic bond, we were able to identify a plausible route (S/H → [H/E] → C) from the reactant to the product at the G(-1) site.
糖苷水解酶（GHs）催化的碳水化合物降解是生物质转化的主要机制。来自 的典型多模块酶家族 9 内切葡聚糖酶（Cel9G），包含一个与家族 3c 碳水化合物结合模块（CBM3c）紧密相连的催化结构域。与其他碳水化合物结合模块提出的常规行为不同，CBM3c 对催化活性有直接影响。在这项工作中，采用广泛的分子动力学（MD）模拟来阐明 CBM3c 的功能作用。此外，使用量子力学和分子力学（QM/MM）方法的组合在原子水平上研究了 Cel9G 的详细催化机制。基于这些模拟，由于肽接头的刚性，CBM3c 可能通过与 GH9 的α螺旋 4 直接相互作用，特别是与 K123 和 H125 残基，影响酶的活性。此外，使用纤维六糖作为底物，QM/MM MD 模拟证实该酶可以通过反转机制切割β-1,4-糖苷键。定位到一个具有 19.6 kcal/mol 势垒高度的氧杂碳正离子样过渡态。此外，在酶-底物复合物中，G(-1)吡喃糖单元优先采用扭曲的 S/H 构象。对于糖苷键的断裂，我们能够在 G(-1)位点从反应物到产物识别出一条合理的途径（S/H → [H/E] → C）。
