YcaO domains use ATP to activate amide backbones during peptide cyclodehydrations

Area of Science:

• Biochemistry and Molecular Biology
• Natural Product Biosynthesis
• Enzymology

Background:

• Thiazole/oxazole-modified microcins (TOMMs) are a class of ribosomally synthesized natural products with varied biological activities.
• The precise mechanism of heterocycle formation by TOMM synthetase enzymes has remained largely elusive despite extensive research.
• Understanding these pathways is crucial for unlocking the potential of these natural products.

Purpose of the Study:

• To elucidate the mechanism of ATP's role in TOMM heterocycle formation.
• To characterize the function of the D-protein component within the TOMM synthetase complex.
• To re-evaluate the classification of enzymes involved in azole and azoline heterocycle installation in ribosomal natural products.

Main Methods:

• Utilized substrate analogs and isotopic labeling to investigate the biochemical pathway.
• Performed enzymatic assays using the D-protein component in isolation.
• Analyzed the role of ATP in the cyclodehydration reaction.

Main Results:

• Demonstrated that ATP directly phosphorylates the peptide amide backbone during TOMM heterocycle formation.
• Provided the first experimental evidence that the D-protein (YcaO/DUF181) alone can perform the ATP-dependent cyclodehydration reaction.
• Showed this reaction occurs independently of other TOMM biosynthetic proteins.

Conclusions:

• ATP is essential for the biosynthesis of azole and azoline heterocycles in ribosomal natural products.
• The D-protein component is the catalytic engine for ATP-dependent heterocycle formation.
• These findings necessitate a reclassification of the enzymes responsible for installing these critical heterocyclic motifs.