Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme

文献类型： 外文期刊

第一作者： Wang, Rong

作者： Wang, Rong;Gong, Li;Xue, Xianli;Qin, Xing;Ma, Rui;Luo, Huiying;Zhang, Yongjie;Yao, Bin;Su, Xiaoyun

作者机构：

期刊名称：PLOS ONE （ 影响因子：3.24； 五年影响因子：3.788 ）

ISSN： 1932-6203

年卷期： 2016 年 11 卷 6 期

页码：

收录情况： SCI

摘要： 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 degrees C) and moreover, was activated by heating at 70 degrees C and 80 degrees 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.

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