Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.
Appl Environ Microbiol. 2021 Feb 12;87(5):e0263420. doi: 10.1128/AEM.02634-20. Epub 2020 Dec 18.
Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, for example, by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is a valuable source for CAZyme and CBM discovery and characterization due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (X181), and a glycoside hydrolase family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, GH5F, is an efficient and specific endo-xyloglucanase (M. A. Attia, C. E. Nelson, W. A. Offen, N. Jain, et al., Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6). In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest products of GH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes. This study reveals carbohydrate-binding module family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the carbohydrate-active enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.
碳水化合物结合模块（CBMs）通常附加在碳水化合物活性酶（CAZymes）上，以增强催化活性，例如增加底物亲和力。革兰氏阴性土壤腐生菌日本纤维弧菌是 CAZymes 和 CBM 发现和表征的有价值的来源，因为它具有内在的能力来降解广泛的植物多糖。对日本纤维弧菌 CJA_2959 基因产物的生物信息学分析显示，其模块化结构由纤维连接蛋白 III （Fn3）模块、一个未知功能的隐模块（X181）和糖苷水解酶家族 5 亚家族 4 （GH5_4）催化模块组成。我们之前证明了最后一个，GH5F，是一种高效且特异性的内切木葡聚糖酶（M. A. Attia、C. E. Nelson、W. A. Offen、N. Jain 等人，Biotechnol Biofuels 11:45, 2018, https://doi.org/10.1186/s13068-018-1039-6）。在本研究中，通过串联超折叠绿色荧光蛋白 C 端融合，实现了在大肠杆菌中的重组生产和纯化。天然亲和凝胶电泳显示出对末端含半乳糖的植物多糖半乳葡甘露聚糖和半乳糖甘露聚糖的结合特异性。等温滴定量热法进一步证明了对半乳葡甘露聚糖多糖的偏好，而不是 GH5F 的限消化产物组成的短寡糖。因此，我们的结果将 X181 模块鉴定为一个新的 CBM 家族，CBM88 的定义成员。除了直接揭示 X181 模块在日本纤维弧菌木葡聚糖代谢中的功能外，本研究还将指导未来微生物（宏）基因组中的生物信息学和功能分析。本研究揭示了碳水化合物结合模块家族 88（CBM88）作为一个新的半乳糖结合蛋白模块家族，该家族与各种微生物糖苷水解酶、多糖裂解酶和碳水化合物酯酶系列存在。在碳水化合物活性酶分类（http://www.cazy.org/CBM88.html）中定义 CBM88 将显著增强未来微生物（宏）基因组分析和功能研究。
