Venditto Immacolata, Luis Ana S, Rydahl Maja, Schückel Julia, Fernandes Vânia O, Vidal-Melgosa Silvia, Bule Pedro, Goyal Arun, Pires Virginia M R, Dourado Catarina G, Ferreira Luís M A, Coutinho Pedro M, Henrissat Bernard, Knox J Paul, Baslé Arnaud, Najmudin Shabir, Gilbert Harry J, Willats William G T, Fontes Carlos M G A
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;
Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):7136-41. doi: 10.1073/pnas.1601558113. Epub 2016 Jun 13.
The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.
植物细胞壁（PCW）聚糖的分解是一个重要的生物学和工业过程。非催化性碳水化合物结合模块（CBMs）在PCW解聚过程中发挥着关键的靶向作用。确定PCW降解系统的CBM组合，即CBMome，对于理解微生物解聚其靶底物的机制至关重要。黄化瘤胃球菌是一种主要的PCW降解细菌，它将其催化装置组装成一个大型多酶复合物——纤维小体。值得注意的是，对黄化瘤胃球菌纤维小体的生物信息学分析未能鉴定出一个预测能与结晶纤维素结合的CBM，而结晶纤维素是其他PCW降解系统的CBMome的一个关键特征。在这里，通过对177个功能未知的蛋白质模块进行高通量筛选，确定了黄化瘤胃球菌的完整CBMome。数据鉴定出了六个以前未鉴定的CBM家族，它们靶向β-葡聚糖、β-甘露聚糖和果胶多糖同型半乳糖醛酸聚糖。四个CBM的晶体结构，结合定点诱变，为配体识别机制提供了见解。在识别β-葡聚糖和β-甘露聚糖的CBM中，保守芳香族残基构象的差异对配体结合裂隙的拓扑结构有显著影响，从而影响配体特异性。CBM77中的一簇碱性残基赋予了对同型半乳糖醛酸聚糖的钙非依赖性识别，表明半乳糖醛酸的羧酸盐是关键的特异性决定因素。本报告表明，黄化瘤胃球菌纤维小体中扩展的蛋白质库有助于形成一个扩展的CBMome，在没有专门靶向结晶纤维素的CBM的情况下支持有效的PCW降解。
