Rational Design of Plant-Derived Protein Ligases with Altered Substrate Specificity.

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• Asparaginyl ligases are enzymes that catalyze site-specific transpeptidation reactions.
• Engineering efforts have improved enzyme efficiency but modifying substrate specificity remains challenging.
• The S2' pocket's tyrosine residue is implicated in substrate specificity.

Purpose of the Study:

• To engineer asparaginyl ligases with altered substrate specificity.
• To investigate the role of the conserved S2' tyrosine residue in substrate specificity.
• To expand the utility of asparaginyl ligases in protein engineering applications.

Main Methods:

• Site-directed mutagenesis of conserved tyrosine residues in asparaginyl ligases (VyPAL2 and butelase 1).
• Assessing the substrate scope of engineered enzymes using various peptide and protein substrates.
• Evaluating the performance of mutant enzymes in peptide cyclization, protein-protein ligation, and N-terminal protein labeling.

Main Results:

• Mutation of the S2' tyrosine residue in VyPAL2 and butelase 1 successfully altered their substrate specificity.
• Engineered ligases demonstrated enhanced processing of substrates for peptide cyclization, protein-protein ligation, and N-terminal protein labeling.
• The conserved S2' tyrosine residue was confirmed as a general determinant of substrate specificity in asparaginyl ligases.

Conclusions:

• The S2' tyrosine residue is a critical determinant for asparaginyl ligase substrate specificity across different plant families.
• Engineering this residue provides a viable strategy for expanding the substrate scope of asparaginyl ligases.
• These findings facilitate the development of more versatile asparaginyl ligases for advanced protein engineering.