Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.
The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.
State Key Laboratory of Microbial Technology, Shandong University , Jinan 250100, China.
Xukai
0000-0002-3624-8732
X
Jiang
State Key Laboratory of Microbial Technology, Shandong University , Jinan 250100, China.
Wen
W
Li
State Key Laboratory of Microbial Technology, Shandong University , Jinan 250100, China.
Guanjun
G
Chen
State Key Laboratory of Microbial Technology, Shandong University , Jinan 250100, China.
Lushan
L
Wang
D020134:Catalytic Domain; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering; D013696:Temperature
D006026:Glycoside Hydrolases