Site-directed mutagenesis (P61G) of copper, zinc superoxide dismutase enhances its kinetic properties and tolerance to inactivation by H2O2

Abstract Superoxide dismutases (SODs) protect the cells by catalyzing the dismutation of harmful superoxide radicals (O2•-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Here, a Cu, Zn SOD (WT) from a high altitude plant (Potentilla atrosanguinea) was engineered by substituting a conserved residue proline to glycine at position 61 (P61G). The computational analysis showed higher structural flexibility and clusters in P61G than WT. The P61G exhibited moderately higher catalytic efficiency (Km = 0.029 μM, Vmax = 1488) than WT protein (Km = 0.038 μM, Vmax = 1290.11). P61G showed higher thermostability as revealed from residual activity (72.25% for P61G than 59.31% for WT), differential calorimetry scanning and CD-spectroscopic analysis. Interestingly, the P61G mutation also resulted in enhanced tolerance to H2O2 inactivation than WT protein. The finding on enhancing the biophysico-chemical properties by mutating conserved residue could stand as an example to engineer other enzymes. Also, the reported mutant can be exploited in food and pharmaceutical industries.