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

To study the molecular basis for thermophilic beta-mannanase of glycoside hydrolase family 5, two beta-mannanases, TlManSA and PMan5A, from Talaromyces leycettanus JCM12802 and Penicillium sp. WN1 were used as models. The four residues, His112 and Phe113, located near the antiparallel beta-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 beta-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 GHS beta-mannanases were elucidated.