A Bifunctional Protease Cleaves the Leader Peptide in the Biosynthesis of Class I Microviridins.

Microviridins are a prominent family of highly potent serine protease inhibitors, of which individual variants specifically inhibit different types of proteases of pharmacological interest. These natural products of cyanobacterial origin belong to the ribosomally synthesized and posttranslationally modified peptides and feature an unusual cage-like architecture, which is composed of characteristic lactone and lactam rings. While the modifying enzymes introducing the posttranslational modifications in the course of microviridin biosynthesis are well investigated, the removal of their N-terminal leader peptides by designated proteases-a key step during maturation-remains enigmatic. In this study, a bioinformatic approach led to the discovery of NosP, which was confirmed as the first specific protease involved in microviridin biosynthesis. In vitro assays with modified precursor peptide, which was obtained from in vitro pathway reconstruction, revealed that NosP is a bifunctional protease, with both endo- and aminopeptidase activities. These results, together with the finding that corresponding homologous leader peptides are widespread in cyanobacteria, may pave the way for the efficient production and bioengineering of class I microviridins in vivo and in vitro.