Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, British Columbia V6T 1Z4, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
J Biol Chem. 2019 Sep 6;294(36):13233-13247. doi: 10.1074/jbc.RA119.009861. Epub 2019 Jul 19.
Glycoside hydrolase family 74 (GH74) is a historically important family of -β-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: , -dissociative, and -processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed β-propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies.
糖苷水解酶家族 74（GH74）是一个历史悠久的β-葡聚糖酶家族。基于早期对纤维素和可溶性纤维素衍生物具有可检测活性的报道，GH74 最初被认为是“纤维素酶”家族，尽管最近的研究普遍表明其对普遍存在的植物细胞壁基质聚糖木葡聚糖具有高度特异性。先前的研究表明，GH74 木葡聚糖酶在骨干切割区域特异性上存在差异，并可以采用三种不同的水解作用模式：非解离、解离和连续。为了提高 GH74 内的功能预测，我们在这里将 17 种重组蛋白的深入生化特性与结构生物学研究相结合，同时构建了一个全面的分子系统发育，包括所有以前表征的家族成员。阐明了四个新的 GH74 三级结构，以及一个来自海洋细菌的远缘双七叶β-螺旋桨蛋白，突出了沿着蛋白质进化轨迹的关键结构-功能关系。我们可以定义五个系统发育组，这些组划定了 GH74 成员的作用方式和区域特异性。在极端情况下，一个主要的酶组分化为非特异性地水解木葡聚糖的骨架，具有解离作用模式和宽松的骨架区域特异性。相比之下，GH74 酶的一个姊妹组进化出了一个由 10 个亚基组成的大型疏水区平台，这有利于连续性。总体而言，我们研究的结果细化了我们对 GH74 催化作用的理解，为未来的实验以及从（宏）基因组研究中出现的序列的生物信息学预测提供了框架。
