Chu Yindi, Tu Tao, Penttinen Leena, Xue Xianli, Wang Xiaoyu, Yi Zhuolin, Gong Li, Rouvinen Juha, Luo Huiying, Hakulinen Nina, Yao Bin, Su Xiaoyun
From the Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
the Department of Chemistry, University of Eastern Finland, Joensuu Campus, Joensuu FIN-80101, Finland.
J Biol Chem. 2017 Nov 24;292(47):19315-19327. doi: 10.1074/jbc.M117.807768. Epub 2017 Oct 3.
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 Xyn10C/Cel48B from 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 Xyn10C 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 Xyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. Xyn10C was found to have a typical (β/α)-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 Xyn10C 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.
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酶促分解中具有节省成本的潜力。来自[具体来源未提及]的双功能多模块酶Xyn10C/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，其底物选择性的分子机制尚未阐明。在此，我们通过对无活性的E140Q/E248Q突变体与木糖和纤维寡糖进行等温滴定量热分析发现，Xyn10C的结合裂隙至少有六个糖结合亚位点。通过测定野生型酶对这些寡糖的催化效率证实了这一点。通过晶体学分析以及分子建模和对接，进一步获得了Xyn10C与木糖或葡萄糖构型的寡糖配体的游离形式和复合物结构。发现Xyn10C具有典型的(β/α)-TIM桶状折叠和GH10酶的“沙拉碗”形状。在与木寡糖的复合物结构中，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，分别有6个和10个氨基酸残基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由-2和-1编号的亚位点上，而发挥中等作用的残基可能位于结合裂隙的更远区域。直接或间接操纵与结合裂隙更远区域底物相互作用的残基可提高Xyn10C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木聚糖酶中是保守的。重新研究随机选择的GH10酶发现了未报道的纤维素酶活性，这表明双功能可能是一种比预期更普遍的现象。
