Improved thermal stability of manganese superoxide dismutase from Staphylococcus equorum through formation of artificial oligomer

Manganese superoxide dismutase (MnSOD) is an important enzyme to remove reactive oxygen species (ROS). It is active as a dimer, but increasing the temperature leads to dimer dissociation, which in turn reduces enzyme activity. In Staphylococcus equorum MnSOD, the dimer dissociates at approximately 55°C, while the monomer unfolds at around 67°C. Previous attempts to strengthen interactions at the dimer interface have typically resulted in reduced enzyme activity and/or reduced stability. Recently, introducing an additional interaction near the interface successfully raised the dimer dissociation temperature. However, since this interaction was non-covalent, the monomers still separated at high temperatures. To prevent dissociation, a covalent bond might be required. Here, we show that introducing intermolecular disulfide bonds by the D47C and D47CE115C mutations promoted oligomer formation. The mutant enzymes exhibited enhanced resistance to dissociation, significantly improved the dimer's thermal stability, and retained enzymatic activity compared to the wild type, maintaining their functional integrity at high temperature, thus paving the way for application of the enzyme in biotechnology and medicine.