Catalytic His-loop flexibility drives high activity in hyperthermophilic esterase EstE1 while preserving structural...

EstE1, a hyperthermophilic esterase, exhibits high catalytic activity despite its rigid structure. This study investigates how the catalytic His loop modulates enzyme activity in EstE1 and its mesophilic counterpart, rPPE. Mutations were introduced at Gly282 in EstE1 (G282N and G282Q) to promote hydrogen bond formation and at Asp287 in rPPE (D287G and D287E) to disrupt or strengthen loop-stabilizing interactions. In EstE1, both G282N and G282Q reduced activity. Wild-type (WT) EstE1 displayed a gradual, temperature-dependent fluorescence decrease from 4°C to 90°C, partly attributed to Tyr182 located in the active-site wall. The G282N mutant exhibited similar fluorescence between 4°C and 25°C and increased rigidity in acrylamide quenching, consistent with hydrogen bond formation, but demonstrated a time-dependent loss in residual activity. In contrast, G282Q failed to form the hydrogen bond and exhibited greater flexibility than WT. In rPPE, increased loop flexibility in D287G enhanced activity without compromising active-site stability. The D287E mutant, which maintained activity similar to WT, formed a stronger hydrogen bond and improved both stability and substrate affinity. These findings suggest that Gly282 promotes His-loop flexibility, which is essential for EstE1's high activity at elevated temperatures, whereas rPPE relies on hydrogen bonding to stabilize the His loop, limiting activity but preserving active-site stability. Overall, this study provides insights into engineering thermophilic enzymes with improved catalytic performance while maintaining structural stability.IMPORTANCEEsterases, which commonly share the catalytic triad Ser-His-Asp, play essential roles in diverse biological processes by catalyzing ester bond formation and cleavage. EstE1 features a flexible catalytic His loop despite its overall rigid structure, whereas its mesophilic counterpart, rPPE, has a more flexible global structure but a rigid His loop stabilized by hydrogen bonding. Introducing a hydrogen bond into the His loop of EstE1 reduces activity and active-site stability, while disrupting this interaction in rPPE enhances activity without compromising stability. Fluorescence and quenching analyses further highlight distinct loop conformations and flexibility profiles among the different mutants. These findings illustrate how EstE1 achieves high activity at elevated temperatures by incorporating catalytic His-loop flexibility into its rigid scaffold. This mechanistic insight provides a valuable framework for engineering thermophilic enzymes with enhanced catalytic performance while preserving structural stability.