Peptide and Protein Cyclization by a Promiscuous Graspetide Synthetase

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Macrocyclic peptides are crucial in drug discovery.
• Graspetides, a type of RiPP, feature macrocycles formed by ATP-grasp enzymes.
• Previous work identified pre-fuscimiditide, a graspetide with a stem and a 10 amino acid loop.

Purpose of the Study:

• To investigate the substrate tolerance and catalytic capabilities of the fuscimiditide ATP-grasp enzyme, ThfB.
• To explore ThfB's potential for diverse macrocyclization and protein cross-linking applications.

Main Methods:

• Enzymatic assays using modified pre-fuscimiditide substrates.
• Testing ThfB's activity with varying stem modifications and loop lengths.
• Evaluating ThfB's ability to cyclize substrates with protein insertions and cross-link protein chains.

Main Results:

• ThfB exhibits high promiscuity, efficiently cyclizing substrates with altered stem regions.
• The enzyme tolerates significant variations in loop length, including glycine-serine sequences from 4 to 72 amino acids.
• ThfB successfully macrocyclized substrates with full-length proteins replacing the native loop and demonstrated intermolecular protein cross-linking capabilities.

Conclusions:

• ThfB is a highly versatile biocatalyst for peptide macrocyclization.
• ThfB's ability extends to protein macrocyclization and intermolecular protein cross-linking.
• The findings suggest ThfB as a powerful tool for engineering novel cyclic peptides and proteins.