Reprogramming nonribosomal peptide synthetases for "clickable" amino acids

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzymology

Background:

• Nonribosomal peptide synthetases (NRPSs) are crucial enzymes for synthesizing diverse bioactive peptides.
• Modifying NRPS specificity is key to expanding their synthetic capabilities.

Purpose of the Study:

• To engineer NRPS enzymes to accept non-natural amino acids containing azide and alkyne functionalities.
• To demonstrate the utility of these modified NRPSs for creating novel peptides.

Main Methods:

• A single tryptophan-to-serine mutation was introduced into phenylalanine-specific NRPS adenylation domains.
• Enzyme activity and substrate specificity were assessed using in vitro and in vivo assays.
• Incorporation of modified amino acids into diketopiperazines was confirmed.

Main Results:

• The mutation resulted in a 10^5-fold switch in substrate specificity, enabling activation of azide- and alkyne-functionalized aromatic amino acids.
• Catalytic efficiency was largely preserved despite the significant specificity change.
• O-propargyl-L-tyrosine was successfully incorporated into diketopiperazines both in vitro and in vivo, even with competing phenylalanine.

Conclusions:

• Reprogramming NRPS specificity via targeted mutations is an effective strategy for incorporating unnatural amino acids.
• The developed method allows for the synthesis of peptides with azide and alkyne groups, facilitating bioorthogonal click chemistry.
• This approach offers a powerful tool for labeling, isolating, and modifying biologically active peptides.