Exploring the Structural Insights of Thermostable Geobacillus esterases by Computational Characterization

This study conducted an in silico analysis of two biochemically characterized thermostable esterases, Est2 and Est3, from Geobacillus strains. To achieve this, the amino acid sequences of Est2 and Est3 were examined to assess their biophysicochemical properties, evolutionary connections, and sequence similarities. Three-dimensional models were constructed and validated through diverse bioinformatics tools. Molecular dynamics (MD) simulation was employed on a pNP-C2 ligand to explore interactions between enzymes and ligand. Biophysicochemical property analysis indicated that aliphatic indices and theoretical T_m values of enzymes were between 82–83 and 55–65 °C, respectively. Molecular phylogeny placed Est2 and Est3 within Family XIII, alongside other Geobacillus esterases. DeepMSA2 revealed that Est2, Est3, and homologous sequences shared 12 conserved residues in their core domain (L39, D50, G53, G55, S57, G92, S94, G96, P108, P184, D193, and H223). BANΔIT analysis indicated that Est2 and Est3 had a significantly more rigid cap domain compared to Est30. Salt bridge analysis revealed that E150–R136, E124–K165, E137–R141, and E154–K157 salt bridges made Est2 and Est3 more stable compared to Est30. MD simulation indicated that Est3 exhibited greater fluctuations in the N-terminal region including conserved F25, cap domain, and C-terminal region, notably including H223, suggesting that these regions might influence esterase catalysis. The common residues in the ligand-binding sites of Est2–Est3 were determined as F25 and L167. The analysis of root mean square fluctuation (RMSF) revealed that region 1, encompassing F25 within the β2–α1 loop of Est3, exhibited higher fluctuations compared to those of Est2. Overall, this study might provide valuable insights for future investigations aimed at improving esterase thermostability and catalytic efficiency, critical industrial traits, through targeted amino acid modifications within the N-terminal region, cap domain, and C-terminal region using rational protein engineering techniques.