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.
• Existing enzymatic methods for CTP synthesis have narrow substrate scopes.

Purpose of the Study:

• To discover novel enzymes for efficient and broad-scope tetrapeptide cyclization using a bioinformatics-guided approach.
• To identify a thioesterase enzyme capable of catalyzing head-to-tail cyclization of tetrapeptides.
• To characterize the substrate scope and mechanism of the discovered enzyme.

Main Methods:

• Bioinformatic analysis of cryptic nonribosomal peptide synthetase gene clusters to predict thioesterase function.
• In vitro characterization of a candidate Penicillin-binding type thioesterase (PBP-TE), designated WP516.
• AlphaFold modeling and covalent docking to elucidate enzyme-substrate interactions and rationalize substrate scope.

Main Results:

• Discovery of WP516, a stand-alone thioesterase enzyme from a cryptic gene cluster.
• WP516 demonstrates efficient cyclization of a broad range of tetrapeptide substrates, significantly exceeding the scope of previously known enzymes like Ulm16.
• Structural modeling provided insights into WP516's broad substrate specificity.

Conclusions:

• The bioinformatics-guided discovery workflow is effective for identifying novel enzymes involved in peptide cyclization.
• WP516 represents a significant advancement in biocatalytic production of cyclic tetrapeptides.
• This work provides a general strategy for discovering enzymes for peptide cyclization, expanding the toolkit for natural product synthesis.