To study the molecular basis for thermophilic β-mannanase of glycoside hydrolase family 5, two β-mannanases, TlMan5A and PMan5A, from Talaromyces leycettanus JCM12802 and Penicillium sp. WN1 were used as models. The four residues, His112 and Phe113, located near the antiparallel β-sheet at the barrel bottom and Leu375 and Ala408 from loop 7 and loop 8 of PMan5A, were inferred to be key thermostability contributors through module substitution, truncation, and site-directed mutagenesis. The effects of these four residues on the thermal properties followed the order H112Y > A408P > L375H > F113Y and were strongly synergetic. These results were interpreted structurally using molecular dynamics (MD) simulations, which showed that improved hydrophobic interactions in the inner wall of the β-barrel and the rigidity of loop 8 were caused by the outside domain of the barrel bottom and proline, respectively. The TIM barrel bottom and four specific residues responsible for the thermostability of GH5 β-mannanases were elucidated.

中文翻译：

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对糖苷水解酶家族5β-甘露聚糖酶的嗜热机理的认识。

为了研究糖苷水解酶家族5的嗜热β-甘露聚糖酶的分子基础，得自Talaromyces leycettanus JCM12802和Penicillium sp。的两种β-甘露聚糖酶，T1 Man5A和P Man5A 。WN1被用作模型。位于桶底部反平行β-折叠附近的四个残基His112和Phe113，以及P的第7环和第8环的Leu375和Ala408通过模块替换，截短和定点诱变，推断Man5A是关键的热稳定性贡献者。这四个残基对热性能的影响遵循H112Y> A408P> L375H> F113Y的顺序，并且具有很强的协同作用。使用分子动力学（MD）模拟从结构上解释了这些结果，结果表明，β-桶内壁中疏水性相互作用的改善和环8的刚度分别是由桶底部和脯氨酸的外域引起的。阐明了TIM桶底部和负责GH5β-甘露聚糖酶热稳定性的四个特定残基。