Improving the catalytic efficiency and dimeric stability of Cu,Zn superoxide dismutase by combining structure-guided consensus approach with site-directed mutagenesis

Superoxide dismutase (SOD) leads the front line of defense against injuries mediated by the reactive oxygen species (ROS). The SOD from a high-altitude plant Potentilla atrosanguinea is a unique thermostable enzyme. In this study, we applied a structure-guided consensus approach on Cu,Zn SOD from Potentilla atrosanguinea plant, to improve its enzymatic properties. The polar uncharged amino acid (threonine) at position 97 of wild-type (WT) SOD was selected as a target residue for substitution by aspartate (T97D) through site-directed mutagenesis. The WT and T97D were examined by a combinative approach consisting of robust computational and experimental tools. The in-silico analysis indicated improved dimeric stability in T97D as compared to the WT. The strong interactions between the monomers were related to improved dimerization and enhanced catalytic efficiency of T97D. These results were validated by in-vitro assays showing improved dimer stability and catalytic efficiency in T97D than WT. Moreover, the mutation also improved the thermostability of the enzyme. The combined structural and functional data described the basis for improved specific activity and thermostability. This study could expand the scope of interface residue to be explored as targets for designing of SODs with improved kinetics.

Automated summary

This study utilized a structure-guided consensus approach to improve the enzymatic properties of Cu,Zn SOD from the high-altitude plant Potentilla atrosanguinea. The T97D substitution in SOD led to improved dimer stability and catalytic efficiency compared to the wild-type (WT), as validated by computational and experimental tools. This mutation not only enhanced enzyme thermostability but also laid the groundwork for designing SODs with improved kinetics by exploring interface residues as potential targets.