Yang Hong, Shi Pengjun, Liu Yun, Xia Wei, Wang Xiaoyu, Cao Huifang, Ma Rui, Luo Huiying, Bai Yingguo, Yao Bin
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, People's Republic of China.
Appl Environ Microbiol. 2017 Mar 2;83(6). doi: 10.1128/AEM.03123-16. Print 2017 Mar 15.
Glycoside hydrolase (GH) family 12 comprises enzymes with a wide range of activities critical for the degradation of lignocellulose. However, the important roles of the loop regions of GH12 enzymes in substrate specificity and catalytic efficiency remain poorly understood. This study examined how the loop 3 region affects the enzymatic properties of GH12 glucanases using EG12A from P1 and EG (PDB 1KS4) from Acidophilic and thermophilic EG12A had the highest catalytic efficiency (/ , 3,001 and 263 ml/mg/s toward lichenin and carboxymethyl cellulose sodium [CMC-Na], respectively) known so far. Based on the multiple-sequence alignment and homology modeling, two specific sequences (FN and STTQA) were identified in the loop 3 region of GH12 endoglucanases from fungi. To determine their functions, these sequences were introduced into EG12A, or the counterpart sequence STTQA was removed from EG. These modifications had no effects on the optimal pH and temperature or substrate specificity but changed the catalytic efficiency (/ ) of these enzymes (in descending order, EG12A [100%], EG12A-FN [140%], and EG12A-STTQA [190%]; EG [100%] and EGΔSTTQA [41%]). Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bond interactions between the substrate and protein, thereby increasing the turnover rate (). This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis. Loop structures play critical roles in the substrate specificity and catalytic hydrolysis of GH12 enzymes. Three typical loops exist in these enzymes. Loops 1 and 2 are recognized as the catalytic loops and are closely related to the substrate specificity and catalytic efficiency. Loop 3 locates in the -1 or +1 subsite and varies a lot in amino acid composition, which may play a role in catalysis. In this study, two GH12 glucanases, EG12A and EG, which were mutated by introducing or deleting partial loop 3 sequences FN and/or STTQA, were selected to identify the function of loop 3. It revealed that the longer loop 3 of GH12 glucanases may strengthen the hydrogen network interactions between the substrate and protein, consequently increasing the turnover rate (). This study proposes a strategy to increase the catalytic efficiency of GH12 glucanases by improving the hydrogen network between substrates and catalytic loops.
糖苷水解酶（GH）家族12包含对木质纤维素降解至关重要的多种活性酶。然而，GH12酶的环区在底物特异性和催化效率方面的重要作用仍知之甚少。本研究使用来自嗜酸性和嗜热菌的P1的EG12A和来自嗜热菌的EG（PDB 1KS4）研究了环3区域如何影响GH12葡聚糖酶的酶学性质。嗜酸性和嗜热的EG12A具有迄今为止已知的最高催化效率（/，分别对地衣多糖和羧甲基纤维素钠[CMC-Na]的催化效率为3,001和263 ml/mg/s）。基于多序列比对和同源建模，在真菌来源的GH12内切葡聚糖酶的环3区域中鉴定出两个特定序列（FN和STTQA）。为了确定它们的功能，将这些序列引入EG12A，或者从EG中去除对应序列STTQA。这些修饰对最佳pH和温度或底物特异性没有影响，但改变了这些酶的催化效率（/）（从高到低依次为：EG12A[100%]、EG12A-FN[140%]和EG12A-STTQA[190%]；EG[100%]和EGΔSTTQA[41%]）。分子对接和动力学模拟分析表明，GH12中较长的环3可能会加强底物与蛋白质之间的氢键相互作用，从而提高周转速率（）。本研究为理解环3对GH12内切葡聚糖酶催化作用的重要作用提供了新的见解。环结构在GH12酶的底物特异性和催化水解中起关键作用。这些酶中存在三个典型的环。环1和环2被认为是催化环，与底物特异性和催化效率密切相关。环3位于-1或+1亚位点，氨基酸组成差异很大，可能在催化中起作用。在本研究中，选择了两个通过引入或删除部分环3序列FN和/或STTQA而发生突变的GH12葡聚糖酶EG12A和EG，以鉴定环3的功能。结果表明，GH12葡聚糖酶中较长的环3可能会加强底物与蛋白质之间的氢键网络相互作用，从而提高周转速率（）。本研究提出了一种通过改善底物与催化环之间的氢键网络来提高GH12葡聚糖酶催化效率的策略。
