Exploring the structure and function of the peptidase-ABC transporter fusion protein ZmaM.

Abstract

Bacteria compete with other microbes for scarce resources through bacterial warfare. One way they accomplish this is by producing and secreting antifungal, antiprotist, and antibiotic compounds. To protect themselves from these toxic compounds, some bacteria produce inactive forms of these molecules and activate them once they are exported. One class of these prodrug molecules is produced with N-acyl-D-Asn prodrug motifs that are then processed by two different kinds of prodrug-activating peptidases. Type I peptidases are serine peptidases composed of a periplasmic S12 hydrolase domain and a three-helix transmembrane domain (TMD). The most well-known type I peptidase is ClbP, which activates the E. coli genotoxin colibactin. Type II peptidases contain an additional ABC half-transporter module fused to the C-terminus of the three-helix TMD. While we are beginning to understand the structure and function of type I peptidases, little is known about type II peptidases. We are investigating the type II peptidase ZmaM, involved in synthesizing the B. cereus antimicrobial zwittermicin, as a model system for understanding the structure and function of these fused proteins. We are pursuing high-resolution structures using cryoEM to understand the overall placement of the two functional modules. Concurrently, we are also using in vitro and in vivo activity assays to evaluate the peptidase, transport and ATPase activities of ZmaM and probing whether any coordination exists between its two functional modules. Understanding the function of this unusual fusion protein gives insight into the production of zwittermicin and other bioactive compounds that use type II peptidases in their synthesis. This work can also facilitate bioengineering studies into modifying ZmaM's activity for zwittermicin production or altering its specificity for designer drugs that benefit from contiguous secretion and processing.