Badino Silke Flindt, Bathke Jenny Kim, Sørensen Trine Holst, Windahl Michael Skovbo, Jensen Kenneth, Peters Günther H J, Borch Kim, Westh Peter
Research Unit for Functional Biomaterials, Department of Science and Environment, INM, Roskilde University, 1 Universitetsvej, Build. 28 C, DK-4000, Roskilde, Denmark.
Department of Chemistry, Technical University of Denmark, Kemitorvet, Build. 207, DK-2800 Kgs. Lyngby, Denmark.
Protein Eng Des Sel. 2017 Jul 1;30(7):495-501. doi: 10.1093/protein/gzx036.
Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type, and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide.
各种纤维素酶由一个催化结构域通过一个柔性连接肽连接到一个碳水化合物结合模块（CBM）组成。连接肽通常高度O-糖基化，长度一般为20 - 50个氨基酸残基。除了连接两个折叠结构域外，连接肽及其聚糖的功能作用已被广泛讨论，但实验证据仍然稀少。研究最多的纤维素降解酶之一是来自嗜热栖热放线菌的多结构域纤维二糖水解酶Cel7A。在此，我们设计了连接肽区域有突变的Cel7A变体，以阐明连接肽的作用。我们发现对连接肽进行适度修饰可能会导致底物亲和力和催化效率发生显著变化。不同的连接肽变体产生的这些变化差异很大。因此，在催化结构域附近缺失六个残基对酶功能基本没有影响。相反，在连接肽中部附近替换四个糖基化位点会降低底物亲和力并提高最大周转率。结合较弱这一观察结果为最近关于连接肽聚糖可能直接参与底物相互作用的观点提供了一些支持。然而，与野生型相比，在CBM附近有几个插入糖基化位点的变体对底物的亲和力也较低，我们认为聚糖与底物的相互作用取决于它们的确切位置以及其他因素，如连接肽结构和动力学的变化。
