Cyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.

Area of Science:

• Biochemistry
• Natural Products Chemistry
• Molecular Biology

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse class of natural products.
• Graspetides are a family of RiPPs characterized by macrocyclization through side chain-side chain ester or amide linkages.
• The ATP-grasp enzyme ThfB was previously identified as the graspetide synthetase responsible for forming ester cross-links in pre-fuscimiditide.

Purpose of the Study:

• To investigate the substrate specificity and catalytic capabilities of the graspetide synthetase ThfB.
• To explore the potential of ThfB in catalyzing alternative cross-linking chemistries beyond ester bonds.
• To develop a novel recombinant methodology for synthesizing head-to-tail cyclic peptides.

Main Methods:

• Enzymatic assays using the graspetide synthetase ThfB with modified peptide substrates.
• Analysis of cross-linking products using mass spectrometry and NMR spectroscopy.
• Proteolysis of thioester-linked peptides followed by native chemical ligation and structural characterization.

Main Results:

• ThfB efficiently catalyzes the formation of thioester cross-links in pre-fuscimiditide, in addition to ester and amide linkages.
• Thioester-linked peptides undergo rapid and quantitative rearrangement into head-to-tail cyclic peptides via native chemical ligation upon N-terminal cysteine exposure.
• The solution structure of the rearranged cyclic peptide was determined, confirming the head-to-tail isopeptide bond.

Conclusions:

• The graspetide synthetase ThfB exhibits broader substrate specificity and catalytic versatility than previously known.
• A novel and efficient recombinant method for producing head-to-tail cyclic peptides has been established.
• This methodology provides a powerful tool for the synthesis of complex cyclic peptides with potential pharmaceutical applications.