The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
复制标题
DOI：
10.1021/ja410291u
复制
发表时间：
2014-01-08
影响因子：
15
通讯作者：
Beckham, Gregg T.
中科院分区：
文献类型：
--
作者：
Knott, Brandon C.;Momeni, Majid Haddad;Beckham, Gregg T.
糖苷水解酶(GHS)通常通过翻转或保留机制断裂碳水化合物中的糖苷键，后者通过两步机制进行，包括形成糖基酶中间体。我们提出了枯草杆菌GH家族7纤维二糖水解酶Cel7A催化结构域的两种新结构，即具有完整的纤维素糖配体和糖基酶中间体的Michaelis复合体，揭示了反应的静态坐标的细节。我们还使用过渡路径采样来确定催化循环的“动态”反应坐标。糖基化反应坐标包含形成和断裂键的成分以及亲核试剂的构象变化。脱糖基化通过产物辅助的机制进行，其中糖基化产物纤维二糖将水分子定位为对糖基酶中间体的异构碳进行亲核攻击。与以前的结构一致，目前的结果揭示了这个自然和工业上重要的酶家族的完整的水解反应配位。
Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.