Activity-guided identification, cloning, heterologous expression, and characterization of a highly thermostable mitochondrial superoxide dismutase from Lantana camara.

Manganese superoxide dismutase (MnSOD), localized in the mitochondrial matrix, is considered pivotal enzyme in the mitochondrial powerhouse. This is primarily due to its central role in the scavenging of excess superoxide anions (O2•-) produced during the electron transport chain. In this study, a highly thermostable MnSOD from Lantana camara (LcMnSOD) was identified via SOD activity assays. Isozyme profiling and thermostability assays demonstrated a high thermal stability of LcMnSOD as it was found to be active even at the temperature exceeding 80 °C. Full-length cDNA (944 bp) encoding LcMnSOD (675 bp) was amplified by rapid amplification of cDNA ends (RACE), followed by recombinant expression in Escherichia coli and further purification via affinity chromatography. The purified enzyme (∼24 kDa) exhibited Km value of 0.026 ± 0.001 μM, Vmax value of 266.29 ± 10.42 Units/mg and was functional across a broad pH (5.0-9.0) and temperature (4-70 °C) range. LcMnSOD existed as a homotetramer and had a high α-helical content, as confirmed by in silico and circular dichroism (CD) analysis. Interestingly, LcMnSOD was resistant to heat inactivation at 80 °C (kd = 0.0063 ± 0.0004 min-1, t1/2 = 116.48 ± 13.96 min), and was also stable to varying concentrations of denaturants, inhibitors, and reducing agents. LcMnSOD could maintain its activity at high temperatures by maintaining a dense hydrogen-bonding network, a robust hydrophobic core, and structural integrity of its catalytic site, ensuring accessibility of substrate-binding residues, as revealed by Molecular dynamics (MD) simulation studies. Owing to its remarkable stability and functional robustness, LcMnSOD holds promise for biotechnological applications.