The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum
The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum
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DOI：
10.1016/j.jbiotec.2013.09.010
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发表时间：
2013-12-01
影响因子：
4.1
通讯作者：
Akhtar, Muhammad Waheed
中科院分区：
文献类型：
--
作者：
Khan, Muhammad Imran M.;Sajjad, Muhammad;Akhtar, Muhammad Waheed
热纤梭菌的木聚糖酶Z以复合物的形式存在于纤维素酶体中，该复合物具有N-末端阿魏酸酯酶、碳水化合物结合模块（CBM 6）和锚定蛋白结构域。为了研究结合模块对XynZ活性的作用，在大肠杆菌中以约30%的总细胞蛋白的水平表达具有在其C-末端（XynZ-CB）和N-末端（XynZ-BC）连接到催化结构域的CBM 6以及在N-末端（XynZ-B 'C）连接的CBM 22的不同变体。XynZ-BC、XynZ-CB和XynZ-B ′ C对桦木木聚糖的活性分别为4200、4180和20，700 U mu M-1。底物结合研究表明，在XynZ-BC和XynZ-CB的情况下，在所用的测定条件下，保持未结合的底物桦木木聚糖分别为51%和52%，而在XynZ-B ′ C的情况下，保持未结合的底物为39%。分子对接研究表明，与XynZ-BC中产生的隧道形状结合口袋相比，XynZ-B 'C中的CBM 22的结合位点更多地暴露，因此可用于底物结合，从而阻碍底物结合。两种构建体的底物结合数据与这一解释一致。(C)2013爱思唯尔有限公司版权所有。
Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B'C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B'C were 4200, 4180 and 20,700 U mu M-1 against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B'C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B'C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation. (C) 2013 Elsevier B.V. All rights reserved.