Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5 β-Mannanase.

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.