S9 protease WprP₂ catalyzes uniform cleavage on the precursor peptide in RiPP biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products synthesized via complex pathways.
• Serine proteases play crucial roles in RiPP biosynthesis by cleaving precursor peptides.
• The diversity of RiPP structures suggests undiscovered enzymes and pathways.

Purpose of the Study:

• To identify and characterize novel serine proteases involved in RiPP biosynthesis.
• To investigate the function of the uncharacterized serine protease WprP2 from Streptomyces venezuelae.
• To expand the understanding of the S9 protease family in natural product synthesis.

Main Methods:

• Bioinformatic analysis to identify potential serine proteases.
• Gene cloning and heterologous expression of WprP2.
• In vitro biochemical assays using precursor peptide WprA2.
• Mass spectrometry to confirm cleavage sites.

Main Results:

• Identified and characterized WprP2, a novel serine protease from Streptomyces venezuelae.
• Demonstrated that WprP2 uniformly cleaves the precursor peptide WprA2 in vitro.
• Observed a unique cleavage activity for WprP2 not previously seen in other serine proteases.
• Confirmed WprP2 is encoded adjacent to a radical S-adenosyl-L-methionine enzyme (WprB2) involved in cyclophane biosynthesis.

Conclusions:

• WprP2 represents a new member of the S9 serine protease family with a distinct role in RiPP biosynthesis.
• The discovery of WprP2 expands the known enzymatic repertoire for RiPP precursor peptide processing.
• This finding contributes to understanding the diversity and mechanisms of natural product biosynthesis.