Recombinant Superoxide dismutase [Mn] (sodA)

Biochemical Characterization

Structure:

• Recombinant MnSOD (sodA) typically forms a homodimer with a subunit molecular mass of ~27 kDa (24 kDa native SOD + 3 kDa His-tag) .

• The active site contains a manganese ion coordinated by four residues: H-27, H-82, D-168, and H-172, with structural domains comprising α-helical and α/β motifs .

• Unlike eukaryotic MnSODs (often tetrameric), prokaryotic variants like Lactococcus lactis MnSOD are dimeric, as confirmed by gel filtration chromatography .

Enzymatic Properties:

• Optimal activity occurs at pH 7.2 and 25°C, with stability up to 45°C .

• Specific activity reaches 18,650 U/mg after purification, as shown below :

• Insensitive to cyanide and hydrogen peroxide, distinguishing it from Cu/Zn-SODs .

Recombinant Production and Purification

Cloning and Expression:

• The sodA gene from L. lactis M4 (621 bp, 206 amino acids) was cloned into pRSET-A and expressed in E. coli BL21(DE3)pLysS using IPTG induction .

• Sequence alignment revealed 98% homology with L. lactis IL1403 sodA, with a single amino acid variation (Tyr202→Asp) .

Purification:

• Immobilized metal affinity chromatography (IMAC) and Superose 12 gel filtration achieved 3.75-fold purification .

• Western blot confirmed successful expression via N-terminal 6xHis-tag fusion .

Functional Roles and Mechanisms

Oxidative Stress Defense:

• Catalyzes dismutation of superoxide radicals ($O_2^−$) into $O_2$ and $H_2O_2$, which are further detoxified by catalases .

• Critical for bacterial survival under oxidative stress; sodA mutants exhibit heightened susceptibility to paraquat and $H_2O_2$ .

Pathogenesis:

• In Streptococcus agalactiae, MnSOD deficiency reduces resistance to macrophage-mediated killing and virulence in murine models .

• Staphylococcus aureus relies on MnSOD (SodA) under manganese-replete conditions, while a cambialistic SOD (SodM) compensates during manganese starvation .

Comparative Analysis with Other SODs

Sources:

Research and Applications

Biotechnological Insights:

• Recombinant MnSOD aids in studying oxidative stress responses in industrial bacteria like L. lactis, enhancing survival in fermentative processes .

• Structural predictions reveal evolutionary conservation of active-site geometry across prokaryotes and eukaryotes .

Therapeutic Potential:

• Human MnSOD analogs show radioprotective and anticancer properties, though prokaryotic sodA is primarily used in mechanistic studies .

• Engineered MnSOD variants could improve microbial robustness in bioremediation or probiotic applications .

Challenges and Future Directions

• Metal Specificity: Strict manganese dependence limits activity under metal starvation, prompting interest in cambialistic SODs (e.g., S. aureus SodM) .

• Structural Optimization: Enhancing thermostability and catalytic efficiency via site-directed mutagenesis remains a focus .