Structure-Guided Engineering of Cu/Zn Superoxide Dismutase for Enhanced Catalytic Efficiency and Thermostability

Cu/Zn superoxide dismutase (Cu/Zn-SOD) is a key antioxidant enzyme that catalyzes superoxide radical dismutation. Here, an integrated computational-assisted, structure-guided engineering strategy was applied to improve Cu/Zn-SOD catalytic performance. Gln22 and Glu134 were identified as promising engineering sites, and saturation mutagenesis yielded Q22I and E134 M as the most beneficial single substitutions, with specific activities 1.55- and 2.39-fold higher than wild type (WT), respectively. Combining both substitutions generated M2 (Q22I/E134M), which showed the highest specific activity of 7121.0 U mg-1, corresponding to a 3.43-fold increase over WT. The optimal temperature of Q22I and M2 increased from 40 to 50 °C, and M2 also exhibited enhanced thermostability. Kinetic analysis showed that M2 had a 41.18% lower Km and a 2.95-fold higher Vmax than WT. Molecular docking suggested complementary effects of Q22I and E134 M on structural stability and active-site optimization, supporting improved catalytic performance. These findings provide a basis for engineering efficient, thermostable SODs.