Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Šresolution is reported that may explain this peculiarity. The crystal belonged to space group P21, with unit-cell parameters a = 61.11, b = 74.55, c = 61.69 Å, ß = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

Introducing Intermolecular Interaction to Strengthen the Stability of MnSOD Dimer.

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity upon treatments like a wide range of pH, addition of detergent and denaturing agent, exposure to ultraviolet light, and heating up to 50 °C. The enzyme dimer dissociates at 52-55 °C, while its monomer unfolds at 63-67 °C. MnSOD dimeric form is indispensable for the enzyme activity; therefore, strengthening the interactions between the monomers is the most preferred strategy to improve the enzyme stability. However, to date, modification of MnSODSeq at the dimer interface has been unfruitful despite excluding the inner and outer sphere regions that are important to the enzyme activity. Here, a new strategy was developed and K38R-A121E/Y double substitutions were proposed. These mutants displayed similar enzyme activity to the wild type. K38R-A121E dimer was thermally more stable and its monomer stability was similar to the wild type. The thermal stability of K38R-A121Y dimer was similar to the wild type but its monomer was thermally less stable. In addition, the structure of the previously reported L169W mutant was also elucidated. The L169W mutant structure showed that intramolecular modification can decrease flexibility of the MnSODSeq monomer and leads to a less stable enzyme with similar activity to the wild type. Thus, while the enzyme activity depends on arrangement of residues in the dimer interface, the stability appears to depend more on its monomeric architecture. Furthermore, in the L169W structure in complex with azide, which is a specific inhibitor for MnSOD, one of the azide molecules was present in the dimer interface region that previously has been identified to involve in the enzymatic reaction. Nevertheless, the present results show that an MnSODSeq mutant with better thermal stability has been obtained.