Engineering the specificity of Streptococcus pyogenes sortase A by loop grafting

Area of Science:

• Microbiology
• Enzymology
• Structural Biology

Background:

• Sortases are Gram-positive bacterial cell-wall enzymes crucial for virulence factor attachment.
• They catalyze transpeptidation reactions by recognizing specific pentapeptide motifs.
• Streptococcus pyogenes sortase A (SpSrtA WT) exhibits flexible substrate specificity, recognizing LPETG, LPETA, and LPKLG motifs.

Purpose of the Study:

• To investigate the role of the β7/β8 loop in SpSrtA's flexible substrate specificity.
• To determine how altering this loop affects sortase activity and substrate recognition.

Main Methods:

• Site-directed mutagenesis: Exchanging the β7/β8 loop of SpSrtA WT with those from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT).
• Enzymatic assays to assess the activity of the modified sortases against different peptide substrates.

Main Results:

• The BaSrtA-derived variant showed no activity against LPETG or LPETA substrates.
• The SaSrtA-derived mutant lost all activity toward the LPETA substrate.
• This mutant exhibited enhanced activity toward the LPETG substrate, which is the preferred substrate for SaSrtA WT.

Conclusions:

• The β7/β8 loop plays a critical role in determining SpSrtA's substrate specificity.
• Modifications to this loop can significantly alter the enzyme's preference for different pentapeptide motifs.
• This finding provides insights into sortase enzyme engineering and understanding bacterial pathogenesis.