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Dramatic Changes in Oligomerization Property Caused by Single Residue Deletion in Staphylococcus aureus Enolase
Vijay Hemmadi1, Malabika Biswas2
1Department of Biological Sciences, Birla Institute of Technology and Science, Pilani, K. K. Birla Goa Campus, NH17B, Zuarinagar, Goa, 403726, India.
Molecular Biotechnology
|January 1, 2021
Summary
C-terminal lysine residues are crucial for Staphylococcus aureus enolase folding and oligomerization. While a K-434Δ mutation slightly reduces catalytic efficiency, the enzyme remains stable and active under various conditions.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Staphylococcus aureus enolase plays a role in bacterial metabolism.
- Enolase exists as a stable dimer and a catalytically active octamer in solution.
- C-terminal lysine residues are investigated for their impact on enzyme function.
Purpose of the Study:
- To elucidate the role of C-terminal lysine residues in Staphylococcus aureus enolase.
- To assess the effects of C-terminal lysine deletion on enzyme kinetics, stability, and oligomeric properties.
- To understand the structural basis of enolase activity and stability.
Main Methods:
- Site-directed mutagenesis to create a K-434Δ deletion mutant.
- Enzyme kinetics assays measuring Km, Vmax, and turnover number.
- Spectroscopic analyses including intrinsic tryptophan fluorescence and circular dichroism (CD) spectroscopy.
- Oligomeric state analysis under varying pH, temperature, and divalent metal ion conditions.
Main Results:
- The K-434Δ mutant showed a minor decrease in Km but a 20-25% reduction in Vmax and a 2% decrease in turnover number, indicating largely preserved enzyme efficiency.
- Both wild-type and mutant enolase exhibited stability in secondary structure and octameric conformation across pH 6-9, temperatures 20-40°C, and in the presence of Mg2+, Zn2+, and Mn2+.
- Spectroscopic data revealed no significant alterations in secondary structure or surface architecture due to the mutation, yet a correlation between decreased catalytic activity and oligomeric stability was observed.
Conclusions:
- C-terminal lysine residues are essential for the proper folding and oligomerization of Staphylococcus aureus enolase.
- The K-434Δ mutation does not abolish enzyme activity but affects catalytic efficiency and oligomeric stability.
- The findings highlight the structural importance of C-terminal lysines for maintaining the functional integrity of S. aureus enolase.
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