CHARACTERIZING THIOESTERASES FOR BIOCATALYTIC PEPTIDE MACROCYCLIZATION

Peptide-based therapeutics have emerged as a crucial class of pharmaceutical molecules, balancing the advantages of small molecules and biologics. Their high potency, selectivity, and minimal immunogenicity make them valuable therapeutic agents. However, linear peptides often suffer from poor stability, low bioavailability, and susceptibility to proteolytic degradation, limiting their clinical utility. Macrocyclization has emerged as a powerful strategy to enhance peptide stability, potency, and membrane permeability by imposing conformational rigidity and eliminating terminal charges. While chemical approaches exist, they frequently require specific amino acid sequences, restricting the diversity of accessible macrocyclic scaffolds.

This work explores biocatalytic strategies for peptide macrocyclization through thioesterase enzymes. Specifically, the characterization of Ulm16 and WP516, penicillin-binding protein-type thioesterases (PBP-TEs) capable of efficiently catalyzing head-to-tail macrolactamization of nonribosomal peptides, including synthetically challenging tetrapeptides. Structural and mutagenesis studies reveal the molecular basis of PBP-TE-catalyzed cyclization, laying the groundwork for future enzyme engineering efforts. This work is further extended to the biocatalytic synthesis of macrocyclic indolylamide peptides, demonstrating that the thioesterase domain from Bulbiferamide biosynthesis (Bulb-TE) can install this challenging linkage under mild conditions.

Collectively, this thesis highlights the power of enzyme-based peptide macrocyclization, expanding the biocatalytic toolkit for generating structurally diverse and pharmaceutically relevant macrocyclic peptides. By characterizing novel thioesterases and developing strategies for their discovery, this work provides a foundation for future advancements in enzyme engineering and biocatalytic peptide synthesis.