Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.
Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.
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从多模块双功能酶中鉴定 CbCel9B/Man5A 的 C 端 GH5 结构域作为第一个具有热激活特性的糖苷水解酶
DOI：
10.1371/journal.pone.0156802
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Su X
中科院分区：
文献类型：
--
作者：
Wang R;Gong L;Xue X;Qin X;Ma R;Luo H;Zhang Y;Yao B;Su X
Caldicellulosiruptor besophila编码至少六种独特的多模块糖苷水解酶，这些糖苷水解酶对于植物细胞壁多糖降解至关重要，每种糖苷水解酶具有两个由两到三个碳水化合物结合模块分开的催化结构域。在这六种酶中，三种具有一个具有相同氨基酸序列的N-或C-末端GH5结构域。尽管有一些关于这些多模块酶的报道，但对保守的GH5结构域如何表现知之甚少，这被认为是由于基因复制而重要的。因此，我们克隆了一个代表性的GH5结构域的C-末端的多模块蛋白，即双功能纤维素酶/甘露聚糖酶CbCel 9 B/Man 5A，这已被报道，并在大肠杆菌中表达。在没有任何附加的CBM的情况下，重组CbMan 5A仍然能够水解具有不同主链连接或侧链修饰的各种甘露聚糖底物。虽然CbMan 5A显示出与CbCel 9 B/Man 5A相同的最适pH，但它具有增加的最适温度（90°C），并且此外，通过在70°C和80°C下加热而活化，这是从未报道过的全长蛋白质的性质。然而，CbMan 5A在甘露聚糖底物上的周转数低于CbCel 9 B/Man 5A。这些数据表明，CbMan 5A和其他结构域进化成单一多肽不是一个简单的组装;相反，一个模块的行为可能会受到全长酶中其他模块的影响。差示扫描量热分析进一步表明，加热CbMan 5A不是一个简单的过渡态过程。据作者所知，CbMan 5A是第一个从多模块双功能酶中鉴定出的具有热活化性质的糖苷水解酶。
Caldicellulosiruptor bescii encodes at least six unique multimodular glycoside hydrolases crucial for plant cell wall polysaccharides degradation, with each having two catalytic domains separated by two to three carbohydrate binding modules. Among the six enzymes, three have one N- or C-terminal GH5 domain with identical amino acid sequences. Despite a few reports on some of these multimodular enzymes, little is known about how the conserved GH5 domains behave, which are believed to be important due to the gene duplication. We thus cloned a representative GH5 domain from the C-terminus of a multimodular protein, i.e. the bifunctional cellulase/mannanase CbCel9B/Man5A which has been reported, and expressed it in Escherichia coli. Without any appending CBMs, the recombinant CbMan5A was still able to hydrolyze a variety of mannan substrates with different backbone linkages or side-chain decorations. While CbMan5A displayed the same pH optimum as CbCel9B/Man5A, it had an increased optimal temperature (90°C) and moreover, was activated by heating at 70°C and 80°C, a property not ever reported for the full-length protein. The turnover numbers of CbMan5A on mannan substrates were, however, lower than those of CbCel9B/Man5A. These data suggested that evolution of CbMan5A and the other domains into a single polypeptide is not a simple assembly; rather, the behavior of one module may be affected by the other ones in the full-length enzyme. The differential scanning calorimetry analysis further indicated that heating CbMan5A was not a simple transition state process. To the best knowledge of the authors, CbMan5A is the first glycoside hydrolase with thermal activation property identified from a multimodular bifunctional enzyme.