Biosciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA.
National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA.
Nat Commun. 2018 Mar 22;9(1):1186. doi: 10.1038/s41467-018-03501-8.
Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure-activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.
糖苷水解酶家族 7 纤维二糖水解酶（GH7 CBHs）在纤维素分解真核生物中催化纤维素的解聚，使它们成为关键的发现和工程目标。然而，这些在工业上重要的纤维素酶仍然缺乏强大的结构-活性关系。在这里，我们比较了里氏木霉（TrCel7A）和青霉（PfCel7A）的 CBH，它们都具有由催化结构域（CD）、碳水化合物结合模块和连接子组成的多模块化结构。我们发现 PfCel7A 在生物质上的性能比 TrCel7A 高出 60%。为了了解每个结构域对这种改进的贡献，我们测量了具有交换亚结构域的 CBH 嵌合体文库的酶活性，证明这种增强主要是由 PfCel7A CD 引起的。我们解析了 PfCel7A CD 的晶体结构，并利用这些信息创建了第二个 TrCel7A CD 突变体文库，确定了一个 TrCel7A 双突变体，其活性与野生型 PfCel7A 相近。总的来说，这些结果揭示了能够实现靶向活性改进的 CBH 区域。
