Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis
Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis
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通过结晶和诱变深入了解影响纤维二糖 2-差向异构酶催化方向的潜在因素
DOI：
10.1107/s205979832001222x
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发表时间：
2020
影响因子：
2.2
通讯作者：
Yang Ruijin
中科院分区：
文献类型：
--
作者：
Feng Yinghui;Hua Xiao;Shen Qiuyun;Matthews Melissa;Zhang Yuzhu;Fisher Andrew J.;Lyu Xiaomei;Yang Ruijin
纤维二糖2-差向异构酶(CE)是公认的一种差向异构酶，因为大多数差向异构酶主要对二糖具有差向异构化活性。近年来，发现了几种具有双功能异构化和异构化活性的化合物。它们可以将乳糖转化为乳果糖，乳果糖是一种高价值的双糖，广泛用于食品和制药行业。然而，决定CES中催化方向的因素仍然不清楚。本研究分别以1.54、2.05和1.80 的分辨率测定了三个新发现的CE：cce(来源于Caldicellulosiruptor charcharolyticus的双功能CE)、StCE(来自嗜热螺旋藻DSM6578的双功能CE)和BtCE(来自B4166的单功能CE)的晶体结构，以寻找它们的单功能/双功能性质的结构线索。对不同的CES、YIHS和甘露糖异构酶活性口袋中的氢键网络的比较分析表明，CSCE中对应于His188的组氨酸是催化异构化所唯一需要的。通过对不同CEs的apo和配体结合结构的比对，发现双功能CEs往往在活性中心周围有更多的柔性环和更大的入口，并且CSCE中的柔性环148-181在配体结合过程中显示出明显的构象变化。推测在配体结合过程中重建的柔性环的分子相互作用有助于激发配体以有利于异构化的方式伸展。对CSCE中柔性环的进一步定点突变分析表明，柔性环的残基组成对异构化没有很大影响，但对异构化有影响。特别是，V177D和I178D突变体的异构化活性比野生型分别提高了50%和80%。这项研究提供了有关CES催化性质的结构特征的新信息，可以用来指导未来的分子修饰。
Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Å resolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148–181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.