Designed Streptococcus pyogenes Sortase A Accepts Branched Amines as Nucleophiles in Sortagging

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Enzymology

Background:

• Sortase-mediated ligation (sortagging) is a crucial tool in biotechnology, typically relying on Staphylococcus aureus sortase A (SaSrtA).
• SaSrtA exhibits strict specificity for N-terminal glycine residues, limiting its application scope.
• There is a need for sortase enzymes with broader substrate specificity and improved activity.

Purpose of the Study:

• To rationally design and engineer Streptococcus pyogenes sortase A (SpSrtA) for enhanced transpeptidase activity.
• To investigate the substrate specificity of the engineered SpSrtA variant.
• To demonstrate the utility of the engineered enzyme in protein backbone cyclization.

Main Methods:

• Rational design principles were applied to engineer SpSrtA.
• Site-directed mutagenesis was used to generate the SpSrtA M3 variant (E189H/V206I/E215A).
• Enzyme kinetics and protein backbone cyclization assays were performed to evaluate M3's activity and specificity.

Main Results:

• The SpSrtA M3 variant exhibited up to a 6.6-fold increase in catalytic efficiency compared to wild-type SpSrtA.
• M3 maintained specificity for N-terminal alanine, glycine, and serine residues, as well as branched primary amines.
• The engineered M3 variant was successfully applied for head-to-tail backbone cyclization of proteins.

Conclusions:

• The rationally designed SpSrtA M3 variant represents a significant improvement in sortase engineering.
• M3 offers enhanced catalytic efficiency while retaining desirable substrate specificities for sortagging applications.
• This engineered sortase provides a valuable tool for advanced protein engineering, including backbone cyclization.