<i>In Vitro</i> Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin’s biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studied in vitro and limited tools exist to measure its activity. Here, we describe the in vitro biochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria