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OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

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Asparaginyl Ligases with Engineered Substrate Specificity for Controlled, Sequential Transpeptidation Reactions.

Yan Zhou¹, Simon J de Veer¹, Tristan J Tyler¹

¹Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland, Brisbane, QLD 4072, Australia.

Journal of the American Chemical Society

|June 11, 2025

Summary

This summary is machine-generated.

Engineered asparaginyl ligases offer new protein engineering possibilities. A single mutation (Y188A) in OaAEP1 creates orthogonal substrate specificity, enabling sequential reactions and novel peptide synthesis.

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Published on: August 1, 2018

Area of Science:

Biochemistry
Enzymology
Protein Engineering

Background:

Transpeptidases are crucial tools in peptide and protein engineering.
Asparaginyl ligases are highly efficient transpeptidases with defined specificities.
Engineered variants with orthogonal specificities are needed for advanced sequential transpeptidation.

Purpose of the Study:

To engineer the asparaginyl ligase OaAEP1 for altered substrate specificity.
To explore the potential of engineered ligases for novel peptide synthesis applications.

Main Methods:

Site-directed mutagenesis was used to introduce amino acid substitutions in OaAEP1.
Enzyme activity assays were performed to assess substrate recognition.
Sequential ligation reactions were conducted for protein labeling and cyclic peptide synthesis.

Main Results:

A single amino acid substitution (Y188A) in OaAEP1 created an orthogonal substrate specificity.
The Y188A mutant recognized a novel substrate sequence, distinct from the parent enzyme.
This engineered ligase enabled controlled sequential reactions, including dual N-/C-terminal protein labeling and one-pot cyclic peptide synthesis.

Conclusions:

The Y188A mutation in OaAEP1 provides a powerful tool for sequential transpeptidation.
Engineered asparaginyl ligases can unlock new strategies for protein modification and peptide synthesis.
The Tyr188 residue appears to be a general determinant of asparaginyl ligase substrate specificity.