Conformational Changes in a Hyperthermostable Glycoside Hydrolase: Enzymatic Activity Is a Consequence of the Loop Dynamics and Protonation Balance

Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan) is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5) was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105). In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

来自岩石热袍菌（Thermotoga petrophila）的内切-β-1,4-甘露聚糖酶（Endo-β-1,4-mannanase，简称TpMan）是一种模块化超嗜热酶，可参与含甘露聚糖多糖的降解过程。这类多糖的降解是诸多工业应用的关键步骤。作为对TpMan持续研究的一部分，本研究针对其糖苷水解酶家族5结构域（GH5 domain，对应重组片段命名为TpManGH5）的酶学特性，开展了pH与温度依赖性的系统表征。结果显示，TpManGH5的酶活性具有显著的pH依赖性，最适催化活性对应的pH为6。研究表明，在pH 8的环境中，TpManGH5分子拥有近乎球形的紧密折叠核心，在溶液中几乎无柔性，其整体尺寸与形态与晶体结构高度吻合。但在酸性条件下，TpManGH5的回转半径显著增大，提示分子发生了构象伸展。此外，在酸性pH环境中，TpManGH5的球状性显著降低，形态不再近似球形，这大概率是由于其Y88至A105残基所在的环区在溶液中更为伸展且柔性更高。此外，分子动力学模拟（molecular dynamics simulations）结果显示，pH变化引发的构象变化并未改变TpManGH5的核心区域，仅上述环区存在高度的构象波动。研究结果还提示，该环区的构象变化可有效促进多糖与酶的结合过程。最后，在pH 6的条件下，相较于20℃时的TpManGH5，65℃下的该酶柔性略有提升。本研究开展的生物物理表征结果与酶活数据高度相关，为阐明TpManGH5的温度与pH依赖性催化活性的结构基础提供了全新视角。同时，本研究鉴定的环区可为具备生物技术应用所需特性的理性分子设计提供关键切入点。