Staphylococcus aureus sortase A exists as a dimeric protein in vitro

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• Sortase A (SrtA) is a transpeptidase enzyme crucial for the virulence of Staphylococcus aureus.
• Understanding the oligomeric state and activity of SrtA is important for developing targeted therapeutics.

Purpose of the Study:

• To directly observe and characterize the self-association behavior of Staphylococcus aureus sortase A (SrtA).
• To investigate the functional implications of SrtA dimerization on enzyme activity.

Main Methods:

• Native polyacrylamide gel electrophoresis (PAGE) and fast protein liquid chromatography (FPLC) for observing SrtA self-association.
• Peptide mass fingerprinting and protein sequencing to confirm the dimeric form.
• Analytical sedimentation equilibrium ultracentrifugation to determine the dissociation constant (Kd) for dimer formation.
• Enzyme kinetic studies to compare the activity of monomeric and dimeric SrtA.

Main Results:

• Direct observation of SrtA dimer formation under native conditions.
• Confirmation of the dimeric state through mass spectrometry and protein sequencing.
• Determination of an apparent Kd for SrtA dimer formation of approximately 55 microM.
• Enzyme kinetic data indicating higher activity of the dimeric form of SrtA compared to the monomeric form.

Conclusions:

• SrtA exists and functions as a dimer, with the dimeric form exhibiting enhanced enzymatic activity.
• The self-association of SrtA is a key aspect of its function and may be a target for novel antibiotic strategies.
• Understanding SrtA dimerization provides insights into microbial physiology and potential therapeutic interventions against Staphylococcus aureus infections.