Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose
Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose
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DOI：
10.1074/jbc.m116.752048
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发表时间：
2016-10-21
影响因子：
4.8
通讯作者：
Iino, Ryota
中科院分区：
文献类型：
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作者：
Nakamura, Akihiko;Tasaki, Tomoyuki;Iino, Ryota
里氏木霉Cel 6A（TrCel 6A）是将结晶纤维素水解成纤维二糖的纤维二糖水解酶。在这里，我们直接观察了结晶纤维素I上的单分子完整的TrCel 6A、分离的催化结构域（CD）、纤维素结合模块（CBM）以及CBM和接头（CBM-接头）的反应循环（结合、表面移动和解离）。CBM-接头的结合速率常数几乎是完整TrCel 6A的一半，而CD和CBM的结合速率常数仅为完整TrCel 6A的十分之一。这些结果表明，糖基化的接头区在很大程度上有助于结晶纤维素上的初始结合。结合后，所有样品都表现出缓慢和快速的解离，这可能是由于纤维素表面的异质性导致的两种不同的结合状态造成的。CBM对高亲和力位点的特异性远高于对低亲和力位点的特异性，而CD则没有，这表明CBM将CD引导至结晶纤维素的疏水表面。在纤维素表面上，完整的分子显示出缓慢的进行性运动（8.8 +/-5.5 nm/s）和快速的扩散运动（30-40 nm/s），而CBM-连接体、CD和无催化活性的全长突变体仅显示出快速的扩散运动。这些结果表明，直接结合和表面扩散有助于寻找纤维素链的水解点。进行性运动的持续时间常数为7.7秒，持续性估计为68 +/- 42。我们的研究结果揭示了每个域在反应循环的基本步骤中的作用，并提供了TrCel 6A在结晶纤维素上进行性运动的第一个直接证据。
Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose I. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 +/- 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 +/- 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.