Directed sortase A evolution for efficient site-specific bioconjugations in organic co-solvents.

Area of Science:

• Biocatalysis and enzyme engineering
• Protein engineering
• Biotechnology

Background:

• Sortase A is a transpeptidase enzyme crucial for bacterial cell wall anchoring.
• Improving sortase A's stability and efficiency in organic solvents is vital for synthetic biology applications.
• Enzyme engineering offers a pathway to enhance enzyme performance under non-native conditions.

Purpose of the Study:

• To engineer sortase A variants with increased resistance and catalytic efficiency in organic co-solvents.
• To investigate the impact of mutations on sortase A activity for peptide and amine conjugation.
• To understand the structural basis for enhanced enzyme stability and function.

Main Methods:

• Directed evolution was employed to generate sortase A variants.
• Enzyme activity and resistance assays were performed in 45% (v/v) dimethyl sulfoxide.
• Molecular dynamics (MD) simulations were used to analyze protein conformational changes.

Main Results:

• Sortase A variants R159G and D165Q/D186G/K196V exhibited 2.2-fold increased resistance and 6.3-fold increased catalytic efficiency in 45% dimethyl sulfoxide.
• The D165Q/D186G/K196V variant showed up to 4.7-fold higher activity for conjugating hydrophobic peptides/amines in co-solvents.
• MD simulations indicated that increased conformational mobilities contribute to the enhanced resistance of the engineered sortase A variants.

Conclusions:

• Directed evolution is an effective strategy for engineering sortase A with improved performance in organic solvents.
• The identified mutations enhance both the stability and catalytic activity of sortase A, broadening its applicability.
• Understanding the role of conformational dynamics provides insights for future enzyme design.