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

Serine proteases in ribosomally synthesized and post-translationally modified peptides (RiPPs) catalyze the cleavage on the precursor peptides in the biosynthesis of RiPP natural products. Here, we identified an uncharacterized serine protease WprP₂ from Streptomyces venezuelae NPDC049867, encoded next to the radical S-adenosyl-L-methionine (SAM) enzyme WprB₂ involved in the biosynthesis of cyclophane natural products. In vitro characterization of S9 protease WprP₂ revealed that the precursor peptide WprA₂ is uniformly cleaved. The cleavage activity of WprP₂ has not been seen in any serine proteases and expands the S9 protease in RiPP biosynthesis.