Wu Xiuyun, Zhang Shu, Zhang Qun, Zhao Yue, Chen Guanjun, Guo Weihua, Wang Lushan
State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, 72 Binhai Rd, Qingdao, 266237, Shandong, China.
College of Marine Science, Shandong University, Weihai, 264209, Shandong, China.
Appl Microbiol Biotechnol. 2020 Oct;104(20):8735-8745. doi: 10.1007/s00253-020-10865-9. Epub 2020 Aug 31.
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.
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.
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.
• 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.
具有高比活性的木聚糖酶在半纤维素生物降解中起关键作用，并且具有重要的工业应用价值。位于活性位点的氨基酸决定了酶的生物学特性。在本研究中，进行了结构生物信息学分析和丙氨酸筛选实验，以探究GH11木聚糖酶活性位点各亚位点氨基酸的作用。
在-2至+1亚位点存在高度保守的氨基酸，且相互作用网络集中在催化位点（E86、E178）附近。然而，相对较远亚位点的氨基酸，尤其是+2和+3亚位点的氨基酸数量较少但种类多样。活性位点结构中的氨基酸丙氨酸取代对催化效率产生了不同影响。有趣的是，+2亚位点的突变体Y180A和+3亚位点的突变体Y96A的酶活性降低了近95%，这表明除了-2和+1亚位点的高度保守残基外，这两个芳香族残基对于底物催化也是必需的。此外，在这两个亚位点，具有不同侧链性质的芳香族氨基酸也影响酶活性。与野生型酶相比，突变体Y180W和Y96W在50℃时的比活性分别提高了6.2%和12.8%。
我们阐明了活性位点中氨基酸与底物之间的相互作用，这将有助于理解酶工程中的蛋白质-配体相互作用。
• GH11家族的木聚糖酶是良好的工业候选酶。• 探究了GH11木聚糖酶活性位点各亚位点残基的作用。• +2和+3亚位点的两个芳香族残基对催化是必需的。• Y180W和Y96W在低温下使酶活性提高了6.2%和12.8%。
