Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose
Insights into the roles of non-catalytic residues in the active site of a GH10 xylanase with activity on cellulose
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深入了解具有纤维素活性的 GH10 木聚糖酶活性位点中非催化残基的作用
DOI：
10.1074/jbc.m117.807768
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发表时间：
2017-11-24
影响因子：
4.8
通讯作者：
Su, Xiaoyun
中科院分区：
文献类型：
--
作者：
Chu, Yindi;Tu, Tao;Su, Xiaoyun
双功能糖苷水解酶在用于生物燃料和生物基化学品的植物细胞壁多糖的酶分解中具有节省成本的潜力。来自 Caldicellulosiruptor bescii 的双功能多模块酶 CbXyn10C/Cel48B 的 N 端 GH10 结构域是一种能够同时降解木聚糖和纤维素的酶。然而，其底物混杂性背后的分子机制尚未阐明。在此，我们通过对木寡糖和纤维寡糖失活的 E140Q/E248Q 突变体进行等温滴定量热分析，发现 CbXyn10C 的结合裂口至少有 6 个糖结合亚位点。通过测定野生型酶对这些寡糖的催化效率证实了这一点。通过晶体学分析和分子建模和对接，进一步获得了带有木糖或葡萄糖配置的寡糖配体的CbXyn10C的游离形式和复合结构。发现 CbXyn10C 具有典型的 (β/α)8-TIM 桶状折叠和 GH10 酶的“沙拉碗”形状。在低聚木糖的复杂结构中，发现了七个糖结合亚位点，并鉴定了许多负责底物相互作用的残基。定点诱变表明6和10个氨基酸残基分别是木聚糖和纤维素水解的关键残基。最重要的残基集中在切割位点附近的子位点-2和-1上，而发挥中等作用的残基可能位于结合裂口的更远端区域。操纵与远端区域中的底物相互作用的残基直接或间接提高了 CbXyn10C 对木聚糖和纤维素的活性。大多数纤维素酶活性的关键残基在 GH10 木聚糖酶中都是保守的。重新审视随机选择的 GH10 酶揭示了未报告的纤维素酶活性，表明双重功能可能是比预期更常见的现象。
Bifunctional glycoside hydrolases have potential for cost-savings in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar-binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cello-oligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (β/α)8–TIM barrel fold and “salad-bowl” shape of GH10 enzymes. In complex structures with xylo-oligosaccharides, seven sugar-binding subsites were found, and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that 6 and 10 amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on subsites −2 and −1 near the cleavage site, whereas residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues interacting with substrates in the distal regions directly or indirectly improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.