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

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Published on: February 5, 2020

Highly Efficient Transpeptidase-Catalyzed Isopeptide Ligation.

Simon J de Veer¹, David J Craik¹, Fabian B H Rehm^1,2

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

Journal of the American Chemical Society

|December 23, 2024

Summary

Engineered asparaginyl ligase OaAEP1 enables direct isopeptide ligation using internal 2,3-diaminopropionic acid residues. This enzyme facilitates diverse peptide modifications and protein fusions under mild conditions, expanding synthetic biology applications.

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Area of Science:

Biochemistry
Synthetic Biology
Enzymology

Background:

Transpeptidases traditionally modify peptide backbones.
Adapting enzymes for side chain modification is challenging, often requiring specific tags or conditions.

Purpose of the Study:

To develop a novel method for site-selective peptide and protein modification using engineered enzymes.
To explore the capabilities of the engineered asparaginyl ligase OaAEP1 for isopeptide ligation.

Main Methods:

Engineering the asparaginyl ligase OaAEP1.
Utilizing 2,3-diaminopropionic acid (Dap) as an internal acceptor residue.
Performing reactions under near-neutral pH without additives.

Main Results:

OaAEP1 catalyzes direct isopeptide ligation with an internal Dap residue, mimicking natural substrates.
Efficient formation of diverse isopeptide-linked products, including cyclic peptides and protein-peptide fusions.
Successful ligation with both L- and D-peptide acceptors, including hybrid bicyclic peptide structures.

Conclusions:

Engineered OaAEP1 provides a versatile and efficient tool for isopeptide bond formation.
The enzyme enables site-selective labeling and construction of complex peptide and protein architectures.
OaAEP1 expands the scope of enzymatic peptide modification to include unnatural amino acids and stereoisomers.