Identification of the C-Terminal GH5 Domain from CbCel9B/Man5A as the First Glycoside Hydrolase with Thermal Activation Property from a Multimodular Bifunctional Enzyme.
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.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.|School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China.
Rong
R
Wang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.|College of Animal Sciences, Zhejiang University, Hangzhou, 310058, P. R. China.
Li
L
Gong
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Xianli
X
Xue
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Xing
X
Qin
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Rui
R
Ma
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Huiying
H
Luo
School of Life Sciences, Shanxi University, Taiyuan, Shanxi, 030006, P. R. China.
Yongjie
Y
Zhang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Bin
B
Yao
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Xiaoyun
X
Su
D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006094:Gram-Positive Bacteria / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity; D013696:Temperature
D006026:Glycoside Hydrolases