Nguyen_2023_Appl.Environ.Microbiol__e0066223

Hydrophobic interactions and hydrogen bonds are 2 types of noncovalent interactions that play distinct roles in the folding and structural stability of proteins. However, the specific roles of these interactions in hydrophobic or hydrophilic environments in alpha/beta-hydrolases are not fully understood. A hyperthermophilic esterase EstE1 in a dimer maintains the C-terminal beta8-alpha9 strand-helix via hydrophobic interactions (Phe276 and Leu299), constituting a closed dimer interface. Moreover, a mesophilic esterase rPPE in a monomer maintains the same strand-helix via a hydrogen bond (Tyr281 and Gln306). Unpaired polar residues (F276Y in EstE1 and Y281A/F and Q306A in rPPE) or reduced hydrophobic interactions (F276A/L299A in EstE1) between the beta8-alpha9 strand-helix decrease thermal stability. EstE1 (F276Y/L299Q) and rPPE WT, both with the beta8-alpha9 hydrogen bond, showed the same thermal stability as EstE1 WT and rPPE (Y281F/Q306L), which possess hydrophobic interactions instead. However, EstE1 (F276Y/L299Q) and rPPE WT exhibited higher enzymatic activity than EstE1 WT and rPPE (Y281F/Q306L), respectively. This suggests that alpha/beta-hydrolases favor the beta8-alpha9 hydrogen bond for catalytic activity in monomers or oligomers. Overall, these findings demonstrate how alpha/beta-hydrolases modulate hydrophobic interactions and hydrogen bonds to adapt to different environments. Both types of interactions contribute equally to thermal stability, but the hydrogen bond is preferred for catalytic activity. IMPORTANCE Esterases hydrolyze short to medium-chain monoesters and contain a catalytic His on a loop between the C-terminal beta8-strand and alpha9-helix. This study explores how hyperthermophilic esterase EstE1 and mesophilic esterase rPPE adapt to different temperatures by utilizing the beta8-alpha9 hydrogen bonds or hydrophobic interactions differently. EstE1 forms a hydrophobic dimer interface, while rPPE forms a monomer stabilized by a hydrogen bond. The study demonstrates that these enzymes stabilize beta8-alpha9 strand-helix differently but achieve similar thermal stability. While the beta8-alpha9 hydrogen bond or hydrophobic interactions contribute equally to thermal stability, the hydrogen bond provides higher activity due to increased catalytic His loop flexibility in both EstE1 and rPPE. These findings reveal how enzymes adapt to extreme environments while maintaining their functions and have implications for engineering enzymes with desired activities and stabilities.