Covalent Leader Peptide Probes Reveal Extra-Cluster Enzymes in Ribosomal Peptide Biosynthesis.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Genome-based analyses have advanced the discovery of ribosomally synthesized and post-translationally modified peptides (RiPPs) pathways.
• However, these methods often miss enzymes encoded outside canonical biosynthetic gene clusters.
• Leader peptides (LPs) are crucial for RiPP enzyme-substrate recognition.

Purpose of the Study:

• To develop a chemical-proteomic strategy for directly identifying LP-binding enzymes from native proteomes.
• To leverage leader peptides (LPs) for substrate-guided discovery of RiPP enzymes.
• To investigate enzyme participation beyond canonical biosynthetic gene clusters.

Main Methods:

• Developed a substrate-guided chemical-proteomic strategy using covalent LP probes.
• Utilized photo-cross-linker-carrying LP probes to engage enzymes at LP-recognition sites.
• Applied the strategy to model systems (LctA-LctM, PatE-LynD) and *Streptomyces sparsogenes*.

Main Results:

• Demonstrated that LP probes maintain enzyme catalytic competence.
• Successfully identified lanthipeptide synthetases encoded outside canonical biosynthetic gene clusters in *Streptomyces sparsogenes*.
• Revealed graded cross-cluster enzymatic activities across multiple RiPP pathways.

Conclusions:

• Established a proof-of-concept platform for proteome-level discovery of LP-binding RiPP enzymes.
• Highlighted cross-cluster enzymatic crosstalk as a mechanism for structural diversification in RiPPs.
• Provided a novel approach to uncover the complete enzymatic machinery of RiPP biosynthesis.