title: Structure features of GH10 xylanase from Caldicellulosiruptor bescii: implication for its thermophilic adaption and substrate binding preference.
abstract: Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys306, Arg314and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try17, Phe20, Phe21, and Phe337may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (Tm) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Yong
authors: Y
authors: Zhang
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Jiao
authors: J
authors: An
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Guangyu
authors: G
authors: Yang
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Xiaofei
authors: X
authors: Zhang
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Yuan
authors: Y
authors: Xie
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors: Liuqing
authors: L
authors: Chen
authors: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China yfeng2009@sjtu.edu.cn.
authors: Yan
authors: Y
authors: Feng
mesh_terms: D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000068536:Bacillota / Q000201:enzymology* / Q000235:genetics; D005965:Glucuronates / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005965:Glucuronates; D009844:Oligosaccharides; D014990:Xylans; C570991:xylooligosaccharide; D043364:Endo-1,4-beta Xylanases
keywords: crystal structure; glycoside hydrolase family 10; thermostability; xylanase