Biotechnologically Relevant Enzymes and Proteins
Biotechnologically Relevant Enzymes and Proteins
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DOI：
10.1007/s00253-014-5748-x
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发表时间：
2014-05
影响因子：
5
通讯作者：
Springer-Verlag;Berlin Heidelberg
中科院分区：
文献类型：
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作者：
Springer-Verlag;Berlin Heidelberg
解凝乳酶类芽孢杆菌B-6产生含有主要木聚糖酶亚基的胞外多酶复合物，命名为Xyn 11 A，其包括属于糖基水解酶家族-11（GH 11）和碳水化合物结合模块家族-36（CBM 36）的两个功能结构域，并具有富含甘氨酸和天冬酰胺的接头（接头）。为了阐明每个功能结构域的作用，构建了重组蛋白XynXL和XynX（分别缺失CBM 36和CBM 36和接头）。它们对可溶性木聚糖的木聚糖酶活性相似，而XynXL对不溶性木聚糖的水解活性降低，而XynX没有木聚糖酶活性。为了确定接头及其相邻区域的重要性，使用圆二色性（CD）光谱和三维（3D）结构分析对XynX进行二级结构比对。类芽孢杆菌属GH 11木聚糖酶中的一个7个氨基酸（NTITIGG）的相邻接头序列高度保守。虽然XynX显示出典型的GH 11木聚糖酶结构，但在β6-和β12-折叠和CD光谱中观察到构象缺口。翻转的Arg 163侧链的亚位点也观察到叠加模型分析后。使用木六糖的对接分析表明，Arg 163侧链的翻转显着影响底物结合的亚位点。为了鉴定与稳定底物结合位点相关的氨基酸，设计了具有延伸的C-末端区域的XynX。至少需要7个氨基酸来恢复底物结合和木聚糖酶活性。这些结果表明，7个氨基酸的邻居Xyn 11 A接头在木聚糖酶结构域的活性和构象稳定性中起着重要作用。
Paenibacillus curdlanolyticusB-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases ofPaenibacillusspecies. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the β6- and β12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain.