Hogg Deborah, Pell Gavin, Dupree Paul, Goubet Florence, Martín-Orúe Susana M, Armand Sylvie, Gilbert Harry J
School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK.
Biochem J. 2003 May 1;371(Pt 3):1027-43. doi: 10.1042/BJ20021860.
beta-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.
β-1,4-甘露聚糖酶（甘露聚糖酶）可水解甘露聚糖和葡甘露聚糖，属于糖苷水解酶家族（GHs）5和26。为了研究GH5和GH26甘露聚糖酶在分子结构和生化特性上是否存在根本差异，从日本纤维弧菌中分离出四个编码这些酶的基因，并对编码的糖苷水解酶进行了表征。这四个基因，即man5A、man5B、man5C和man26B，分别编码甘露聚糖酶Man5A、Man5B、Man5C和Man26B。Man26B由一个N端信号肽组成，该信号肽通过一个富含丝氨酸的延伸区域与一个GH26催化结构域相连。Man5A、Man5B和Man5C包含GH5催化结构域和属于2a、5和10家族的非催化碳水化合物结合模块（CBMs）；Man5C还包含一个功能未知的定义为X4的模块。10家族和2a家族的CBMs与结晶纤维素和象牙果结晶甘露聚糖结合，显示出与来自纤维弧菌纤维素酶和木聚糖酶的相应10家族和2a家族CBMs非常相似的特性。CBM5与这些结晶多糖的结合较弱。Man5A、Man5B和Man26B的催化结构域可水解半乳甘露聚糖和葡甘露聚糖，但对结晶甘露聚糖或纤维素底物无活性。虽然Man5C对半乳甘露聚糖和葡甘露聚糖的活性低于其他甘露聚糖酶，但它确实能攻击结晶象牙果甘露聚糖。所有酶在反应初始阶段均表现出典型的内切活性，产生寡糖混合物，尽管它们对甘露寡糖和葡甘露聚糖的作用模式表明各自底物结合位点的拓扑结构存在差异。本报告指出了来自日本纤维弧菌的GH5和GH26甘露聚糖酶的不同作用。我们认为，由于GH5酶含有与结晶多糖结合的CBMs，这些酶可能靶向植物细胞壁中不可或缺的甘露聚糖，而缺乏CBMs且能迅速从多糖和寡糖中释放甘露糖的GH26甘露聚糖酶则靶向储存多糖半乳甘露聚糖和甘露寡糖。
