The contribution of specific subsites to catalytic activities in active site architecture of a GH11 xylanase
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
The contribution of specific subsites to catalytic activities in active site architecture of a GH11 xylanase Xiuyun Wu 1 & Shu Zhang 1 & Qun Zhang 1 & Yue Zhao 1 & Guanjun Chen 1,2 & Weihua Guo 3 & Lushan Wang 1 Received: 29 May 2020 / Revised: 11 August 2020 / Accepted: 26 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Background Xylanase with high specific activity plays a crucial role in hemicellulose biodegradation and has important industrial application. The amino acids located in the active site determine the enzyme biological characterization. In this study, structure bioinformatics analysis and alanine screening experiments were performed to explore the roles of amino acids at each subsite of the GH11 xylanase active site. Results There are highly conserved amino acids at − 2 to + 1 subsites, and the network of the interactions is concentrated near the catalytic sites (E86, E178). However, the amino acids at relatively distal subsites, especially at the + 2 and + 3 subsites, are few but diverse. Alanine substitution of amino acids in the active site architecture exerted different impacts on catalytic efficiency. Interestingly, mutants Y180A at the + 2 subsite and Y96A at the + 3 subsite had reduced enzymatic activities by almost 95%, which indicate that these two aromatic residues are necessary for the catalysis of substrates in addition to the highly conserved residues at the − 2 and + 1 subsites. Moreover, in these two subsites, aromatic amino acids with different side-chain properties also affected enzyme activity. The mutants Y180W and Y96W showed 6.2% and 12.8% increase in specific activities by comparison with wild-type enzyme at 50 °C, respectively. Conclusion We elucidated the interaction between amino acids and substrates in the active site, which will aid understanding of the protein-ligand interaction in enzyme engineering. Key points • Xylanase of GH11 family is a good industrial candidate. • The roles of residues at each subsite of GH11 xylanase active site are explored. • The two aromatic residues at the + 2 and + 3 subsites are necessary for the catalysis. • Y180W and Y96W increased the enzymatic activity by 6.2% and 12.8% at low temperature. Keywords GH11 xylanase . Active site architecture . Enzyme-ligand interaction . Catalytic activities . Site-directed mutagenesis
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10865-9) contains supplementary material, which is available to authorized users. * Lushan Wang [email protected] 1
State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, 72 Binhai Rd, Qingdao 266237, Shandong, China
2
College of Marine Science, Shandong University, Weihai 264209, Shandong, China
3
School of Life Sciences, Shandong University, Qingdao 266237, Shandong, China
Hemicellulose xylan is the second most abundant plant structural polysaccharide, which forms biomass
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