Mutation T71R enhanced the structural stability and functional activity of wild type superoxide dismutase cloned from...

In this study, we report engineering of three mutations m1, m2, and m3 respectively, in the wild type SOD, cloned form soil metagenome. Expressed proteins from wild type and mutants were purified to homogeneity using Ni-NTA affinity chromatography. Biochemical characterization of mutants demonstrated enhanced functional activity at varying pH and temperature compared to wild type and other mutant proteins. Additionally, it also showed increased specific activity of 185 ± 0.75 U/mg compared to 150 ± 0.042 U/mg and 168 ± 0.25 U/mg respectively for mutant m1, m2 and m3. Altogether, it was observed that the relative enzyme activity of mutant m1, m2 and m3 enhanced ∼ 30 %, 10 % and 17 % respectively compared to wild type. Biophysical investigation carried out employing circular dichroism and intrinsic tryptophan fluorescence also demonstrated conformational stability in the secondary and tertiary structure of mutant m1 compared to the wild type at varying pH and temperature. Interestingly, in silico molecular simulation dynamics studies carried out at 300 ns demonstrated structural stability, reduced flexibility and attainment of stable conformation in this mutant form. Molecular simulation analysis revealed that mutation T71R in m1 tends to introduce β-sheet like secondary structure at protein surface, which might enhance residue-residue interactions within this protein, leading to allover enhancement in the stability and activity of this mutant.