Biocatalytic Tetrapeptide Macrocyclization by Cryptic Penicillin-Binding Protein-Type Thioesterases

Area of Science:

• Biochemistry
• Bioinformatics
• Natural Product Synthesis

Background:

• Cyclic tetrapeptides (CTPs) are valuable natural products with diverse bioactivities.
• Synthesizing CTPs is challenging due to ring strain and limitations of current chemical methods.
• Enzymatic approaches for CTP synthesis are sought after for broader substrate scope.

Purpose of the Study:

• To discover novel enzymes for efficient tetrapeptide cyclization using a bioinformatics-guided approach.
• To identify and characterize a thioesterase enzyme capable of producing diverse cyclic tetrapeptides.
• To understand the mechanism behind the enzyme's broad substrate specificity.

Main Methods:

• Bioinformatic analysis of cryptic nonribosomal peptide synthetase (NRPS) gene clusters.
• In silico prediction and identification of penicillin-binding type thioesterases (PBP-TEs).
• Biochemical characterization of the identified enzyme (WP516) with various tetrapeptide substrates.
• Structural and computational analyses including AlphaFold modeling, covalent docking, molecular dynamics, and mutational studies.

Main Results:

• The first bioinformatics-guided discovery of a thioesterase (WP516) for peptide cyclization from a cryptic gene cluster.
• WP516 demonstrates efficient cyclization of a wide range of tetrapeptide substrates.
• WP516 exhibits a significantly broader substrate scope compared to the previously known tetrapeptide cyclase Ulm16.
• Structural and computational analyses provided insights into WP516's broad substrate acceptance.

Conclusions:

• The developed bioinformatics workflow is effective for discovering novel enzymes involved in peptide cyclization.
• WP516 is a powerful biocatalyst for the production of diverse head-to-tail cyclic tetrapeptides.
• This discovery offers a new strategy for enzyme discovery in peptide cyclization and biocatalytic CTP production.