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.

中文翻译：

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特定亚位点对GH11木聚糖酶活性位点构型中催化活性的贡献。

背景技术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，并具有重要的工业应用。位于活性位点的氨基酸决定了酶的生物学特性。在这项研究中，进行了结构生物信息学分析和丙氨酸筛选实验，以探索氨基酸在GH11木聚糖酶活性位点每个子位点的作用。结果在-2至+ 1个亚位点有高度保守的氨基酸，相互作用的网络集中在催化位点附近（E86，E178）。但是，在相对较远的亚位点，尤其是在+ 2和+3亚位点的氨基酸很少，但种类繁多。活性位点结构中氨基酸的丙氨酸取代对催化效率产生不同的影响。有趣的是 + 2子位点的突变体Y180A和+ 3子位点的Y96A突变体的酶活性降低了近95％，这表明除了-2和+处的高度保守残基外，这两个芳族残基对于底物的催化也是必需的1个子站点。而且，在这两个亚位中，具有不同侧链性质的芳族氨基酸也影响酶的活性。与野生型酶在50°C相比，突变体Y180W和Y96W的比活分别提高了6.2％和12.8％。结论我们阐明了氨基酸和活性位点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。要点•GH11家族的木聚糖酶是很好的工业候选者。•探索了GH11木聚糖酶活性位点每个亚位点残基的作用。•+ 2和+ 3亚位的两个芳族残基对于催化是必需的。•在低温下，Y180W和Y96W使酶活性增加了6.2％和12.8％。