Structural basis of colibactin activation by the ClbP peptidase.

Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

A small molecule inhibitor prevents gut bacterial genotoxin production.

The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin's biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis.

Structure and function of prodrug-activating peptidases.

Bacteria protect themselves from the toxicity of antimicrobial metabolites they produce through several strategies. In one resistance mechanism, bacteria assemble a non-toxic precursor on an N-acyl-d-asparagine prodrug motif in the cytoplasm, then export it to the periplasm where a dedicated d-amino peptidase hydrolyzes the prodrug motif. These prodrug-activating peptidases contain an N-terminal periplasmic S12 hydrolase domain and C-terminal transmembrane domains (TMDs) of varying lengths: type I peptidases contain three transmembrane helices, and type II peptidases have an additional C-terminal ABC half-transporter. We review studies which have addressed the role of the TMD in function, the substrate specificity, and the biological assembly of ClbP, the type I peptidase that activates colibactin. We use modeling and sequence analyses to extend those insights to other prodrug-activating peptidases and ClbP-like proteins which are not part of prodrug resistance gene clusters. These ClbP-like proteins may play roles in the biosynthesis or degradation of other natural products, including antibiotics, may adopt different TMD folds, and have different substrate specificity compared to prodrug-activating homologs. Finally, we review the data supporting the long-standing hypothesis that ClbP interacts with transporters in the cell and that this association is important for the export of other natural products. Future investigations of this hypothesis as well as of the structure and function of type II peptidases will provide a complete account of the role of prodrug-activating peptidases in the activation and secretion of bacterial toxins.