Synergistic Enhancement of Thermal Stability and Activity of Glycolaldehyde Synthase via Computer-Aided Design and Mechanistic Analysis.

Efficient C1 utilization relies on glycolaldehyde (GALD) as a key intermediate, typically formed via condensation of two formaldehyde (FALD) molecules catalyzed by glycolaldehyde synthase (GALS). While catalytic activity improvements have been achieved, limited attention to thermal stability has constrained industrial application. Building on the high catalytic efficiency (153.3 M-1·s-1) variant GALS M3 (T87A/A416T/W463I), we applied computer-aided design strategy to further enhance enzyme stability. After two rounds of systematic protein engineering, variant GALS M5 (GALS M3-A381P/K290P) was obtained, which exhibited enhanced thermostability (Tm = 63 °C) while maintaining a high initial activity (2204.55 U/g). This variant represents the highest activity and thermostable GALS reported to date. Additionally, mutant GALS M3-S61A displayed heat-activated behavior, showing a 1.4-fold activity increase after incubation at 50 °C for 3 h. Structural and MD simulation analyses revealed that the stabilizing mutations reinforce loop rigidity in GALS M5, whereas S61A induces conformational rearrangement rather than unfolding at elevated temperatures, conferring its heat activation behavior.