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CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.