Thermodynamics of a Hyperthermostable Carboxylesterase from Anoxybacillus geothermalis D9.

Hyperthermostable enzymes are very attractive biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme displayed remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new β-sheets at 80 °C that make up the core of the hydrolase fold. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, residing at the interface between the two domains. To further investigate molecular adaptation in extreme conditions, the intramolecular interactions of native structure were investigated. 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters were revealed within EstD9, which is higher than the reported thermostable carboxylesterase Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to fundamental and applied research involving protein engineering of industrial thermostable enzymes.