Introducing Intermolecular Interaction to Strengthen the Stability of MnSOD Dimer
Debbie S Retnoningrum1, Hiromi Yoshida2, Ismiana Pajatiwi1
1Laboratory of Pharmaceutical Biotechnology, Pharmaceutics Research Group, School of Pharmacy, Institut Teknologi Bandung, Ganesha 10, Bandung, 40132, West Java, Indonesia.
Applied Biochemistry and Biotechnology
|January 26, 2023
Summary
This study engineered manganese superoxide dismutase (MnSODSeq) mutants to enhance thermal stability. A novel strategy yielded a mutant with improved stability while maintaining enzyme activity, suggesting monomeric architecture is key to stability.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) exhibits remarkable stability across various conditions but dissociates and unfolds at elevated temperatures.
- The dimeric form of MnSOD is crucial for its activity, making monomer interaction enhancement a target for improving enzyme stability.
- Previous attempts to modify the dimer interface of MnSODSeq for stability have been unsuccessful.
Purpose of the Study:
- To develop a new strategy for enhancing the thermal stability of MnSODSeq.
- To investigate the impact of specific mutations (K38R-A121E/Y) on MnSODSeq activity and stability.
- To elucidate the structural basis of MnSODSeq stability and activity, particularly concerning monomeric architecture versus dimer interface.
Main Methods:
- Site-directed mutagenesis was employed to create K38R-A121E and K38R-A121Y double mutants of MnSODSeq.
- Enzyme activity assays were performed to compare mutant activity with the wild type.
- Thermal stability assessments (dimer and monomer) were conducted for the wild type and mutants.
- X-ray crystallography was used to determine the structure of the L169W mutant, including its complex with azide.
Main Results:
- The K38R-A121E mutant showed enhanced thermal stability of the dimer, with similar monomer stability to the wild type, while retaining comparable enzyme activity.
- The K38R-A121Y mutant exhibited similar dimer stability to the wild type but reduced monomer stability.
- Structural analysis of the L169W mutant indicated that intramolecular modifications can decrease monomer flexibility, leading to reduced overall enzyme stability.
- Azide, an MnSOD inhibitor, was observed at the dimer interface in the L169W mutant structure.
Conclusions:
- Enzyme activity of MnSODSeq is dependent on the arrangement of residues at the dimer interface.
- Enzyme stability appears to be more influenced by the monomeric architecture than the dimer interface.
- A strategy involving specific double substitutions successfully yielded an MnSODSeq mutant with improved thermal stability.
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