Tertiary amide bond formation by an engineered asparaginyl ligase

Area of Science:

• Biochemistry
• Chemical Biology
• Protein Engineering

Background:

• Transpeptidases are crucial for site-specific protein modification, enabling the creation of custom biologics.
• Current methods using transpeptidases are limited to forming secondary amide bonds at protein termini.
• Developing new methods for protein modification is essential for advancing therapeutics and diagnostics.

Purpose of the Study:

• To investigate the capability of an engineered asparaginyl ligase to form tertiary amide bonds.
• To explore the potential of this ligase for sequential and dual protein labeling.
• To expand the chemical diversity of protein modification reactions.

Main Methods:

• Utilized an engineered asparaginyl ligase for protein ligation reactions.
• Employed diverse secondary amine nucleophiles to synthesize tertiary amide bonds.
• Investigated the enzyme's recognition elements (P1 Asn and P2'' Leu) and substrate tolerance.
• Incorporated 4-azidoproline for one-pot dual labeling.

Main Results:

• The engineered asparaginyl ligase efficiently synthesized tertiary amide bonds under mild, near-neutral pH conditions.
• The enzyme's optimal recognition elements were preserved during the reaction.
• Proline-containing products showed resistance to enzyme recognition, facilitating sequential labeling.
• One-pot dual labeling was achieved using 4-azidoproline at the ligation junction.

Conclusions:

• Engineered asparaginyl ligases offer a novel route to synthesize tertiary amide bonds, overcoming limitations of existing transpeptidases.
• This method provides a versatile platform for straightforward, successive protein modification.
• The findings expand the toolkit for creating tailored biologics, therapeutics, and diagnostics.