Diverse thioether macrocyclized peptides through a radical SAM maturase

Area of Science:

• Biochemistry
• Chemical Biology
• Biotechnology

Background:

• Disulfide bonds stabilize peptides but are unstable under physiological conditions, limiting biotechnological applications.
• PapB is an S-adenosyl-L-methionine enzyme that typically forms thioether linkages in peptide maturation.
• Ribosomally produced and post-translationally modified peptides require stabilization strategies.

Purpose of the Study:

• To investigate the substrate scope and enzymatic activity of PapB beyond its known function.
• To explore the potential of PapB in forming novel thioether macrocycles using various amino acid substrates.
• To expand the enzymatic toolbox for creating conformationally constrained peptides.

Main Methods:

• Enzymatic assays using PapB with various peptide substrates, including those with D- and β-amino acids.
• Analysis of peptide products to confirm the formation of α- and β-thioether macrocycles.
• Characterization of PapB's tolerance for unnatural amino acids within peptide substrates.

Main Results:

• PapB efficiently couples internal Cys thiols to C-terminal carboxylates of peptides ending in D- or β-amino acids, forming α- or β-thioether macrocycles.
• PapB demonstrates tolerance for β- and N-methyl amino acids, enabling the synthesis of macrocycles composed entirely of unnatural amino acids.
• PapB functions as a sequence-agnostic thioether ligase for C-terminal macrocyclization.

Conclusions:

• PapB is a versatile enzyme capable of forming stable thioether macrocycles with diverse peptide substrates, including unnatural amino acids.
• The findings establish PapB as a valuable tool for constructing conformationally constrained peptides.
• This research broadens the possibilities for peptide-based therapeutics and chemical biology.