Structure of Bacillus cereus ZmaM, a peptidase-ABC transporter that secretes and activates zwittermicin A

Bacteria engage in microbial warfare by secreting toxic molecules. These toxins can serve as therapeutics or drug discovery starting points, motivating investigations of their production. Bacteria that produce these toxins must protect themselves, and a common strategy is to synthesize an inactive prodrug that is activated upon secretion. Some prodrugs are capped with N -acyl-ᴅ-asparagine motifs, which are removed by two types of prodrug peptidases. Type I peptidases are membrane-bound S12 hydrolases; type II peptidases have an additional C-terminal half-ABC transporter module. Recent structures of a type I peptidase revealed how its transmembrane domain completes the substrate-binding site, but little is known about type II peptidases. Here, we show that the B. cereus type II peptidase ZmaM is responsible for both the export of prezwittermicin and its activation into zwittermicin. The structure of homodimeric ZmaM reveals a central type IV ABC transporter flanked by peptidase modules. The periplasmic hydrolase domains interact over the transporter's open outer vestibule. Mutations to intermodule interfaces impact both peptidase and ATP hydrolysis activities, indicating the peptidase and transporter modules are interdependent. Together, our findings reveal an evolutionary strategy to coordinate toxin activation with its export and inform future efforts to harness these systems in biotechnology.