Single mutation on the surface of Staphylococcus aureus Sortase A can disrupt its dimerization

Area of Science:

• Microbiology
• Enzymology
• Structural Biology

Background:

• Staphylococcus aureus Sortase A (SrtA) is a crucial Gram-positive membrane enzyme responsible for anchoring cell surface proteins via the LPXTG motif.
• SrtA has been previously identified as a dimer in vitro with a dissociation constant (Kd) of 55 microM.

Purpose of the Study:

• To investigate the role of SrtA dimerization in its function.
• To identify specific amino acid residues critical for SrtA self-association.
• To characterize the impact of disrupting SrtA dimerization on enzyme activity and structure.

Main Methods:

• Site-directed mutagenesis to introduce single point mutations on the SrtA surface.
• Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography to assess dimer-monomer equilibrium.
• Circular dichroism spectroscopy to analyze conformational changes in SrtA mutants.
• Enzyme activity assays to confirm the functionality of SrtA mutants.

Main Results:

• A single surface point mutation was sufficient to completely abolish SrtA dimerization.
• Native PAGE and gel filtration confirmed the disruption of dimer-monomer equilibrium in mutants.
• Circular dichroism revealed no significant conformational changes in the active SrtA mutants.
• All generated SrtA mutants retained enzymatic activity in vitro.

Conclusions:

• SrtA dimerization is sensitive to specific surface amino acid alterations.
• Disruption of SrtA dimerization does not necessarily abolish its enzymatic activity.
• These findings offer critical insights into the SrtA self-association mechanism and provide tools for in vivo studies.
• The study highlights potential avenues for developing novel Gram-positive antibiotics targeting the sortase A pathway.