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Insight into the potential factors influencing the catalytic direction in cellobiose 2‐epimerase by crystallization and mutagenesis
Acta Crystallographica Section D ( IF 2.2 ) Pub Date : 2020-11-02 , DOI: 10.1107/s205979832001222x
Yinghui Feng 1 , Xiao Hua 1 , Qiuyun Shen 1 , Melissa Matthews 2 , Yuzhu Zhang 3 , Andrew J Fisher 2 , Xiaomei Lyu 1 , Ruijin Yang 1
Affiliation  

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.

中文翻译:

通过结晶和诱变洞察影响纤维二糖2-表异构酶催化方向的潜在因素

纤维二糖2-表异构酶(CE)通常被认为是一种差向异构酶,因为大多数CE主要表现出对二糖的差向异构活性。近年来,已经发现几种CE具有双功能差向异构和异构化活性。它们可以将乳糖转化为乳果糖,乳果糖是一种高价值的二糖,广泛用于食品和制药行业。但是,决定CEs催化方向的因素仍不清楚。在这项研究中,三个新发现的CE的晶体结构为Cs CE(一种来自Caldicellulosiruptor saccharolyticus的双功能CE ),St CE(一种来自Spirochaeta thermophila DSM 6578的双功能CE )和Bt CE(一种来自CE的单功能CE)。为了寻找其单功能/双功能性质的结构线索,分别以1.54、2.05和1.80的分辨率测定了热芽孢杆菌B4166)。对不同CE,YihS和甘露糖异构酶活性口袋中氢键网络的比较分析表明,Cs CE中与His188对应的组氨酸是催化异构化的唯一条件。通过排列不同CEs的载脂蛋白和配体结合结构,发现双功能CEs趋向于具有更多的柔性环和活性位点附近更大的入口,并且Cs中的柔性环148–181CE在配体结合过程中显示出明显的构象变化。据推测,在配体结合期间柔性环的重构分子相互作用有助于以有利于异构化的方式激励配体拉伸。对Cs CE中柔性环的进一步定点诱变分析表明,柔性环的残基组成不会极大地影响差向异构,但会影响异构化。特别是,V177D和I178D突变体的异构化活性比野生型提高了50%和80%。这项研究提供了有关CEs催化特性中涉及的结构特征的新信息,可用于指导未来的分子修饰。
更新日期:2020-11-02
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