An Engineered Biarylitide Cross-Linking P450 from RiPP Biosynthesis Generates Alternative Cyclic Peptides

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Peptide Chemistry

Background:

• Cytochrome P450 enzymes catalyze cross-linking reactions in ribosomally encoded peptides (RiPPs).
• This P450-mediated RiPP cross-linking is a growing field with significant biocatalytic potential.
• Existing methods primarily focus on specific cross-linking motifs.

Purpose of the Study:

• To engineer the active site of the biarylitide cross-linking enzyme P450_Blt.
• To expand the substrate scope of P450-mediated peptide cross-linking.
• To demonstrate the production of novel cyclic tripeptides with controlled cross-linking.

Main Methods:

• Site-directed mutagenesis of the P450_Blt enzyme's active site.
• In vitro assays to assess cross-linking activity on synthetic peptide substrates.
• Analysis of peptide products using mass spectrometry and other biochemical techniques.

Main Results:

• Successfully engineered P450_Blt to form His-X-Tyr cross-linked peptides.
• Demonstrated the formation of Tyr-X-Tyr cross-linked peptides using the engineered enzyme.
• Achieved controlled cross-linking states in the resulting cyclic tripeptides.

Conclusions:

• Active site engineering of P450_Blt provides a versatile platform for generating diverse cyclic tripeptides.
• This approach broadens the utility of cytochrome P450 enzymes in biocatalysis and peptide synthesis.
• The findings pave the way for novel peptide-based therapeutics and materials.