J. Am. Chem. Soc. doi:10.1021/jacs.7b09971 (2017)

The natural product colibactin, biosynthesized by strains of Escherichia coli linked to colorectal cancer, contains an electrophilic cyclopropane moiety that is critical for its genotoxic activity in vitro. E. coli use several self-immunity strategies to protect themselves against the activity of colibactin, among them the resistance protein ClbS. Tripathi et al. have now determined that ClbS provides this protection by catalyzing hydrolysis of colibactin's cyclopropane warhead. ClbS exhibits selectivity for electrophilic colibactin analogs, as it does not hydrolyze an alternatively cyclized and stable aromatic scaffold. Although ClbS was initially identified through labeling with an activity-based probe, the crystal structure of ClbS revealed that the cysteine residue alkylated by the probe is not actually located in the active site of the enzyme and is dispensable for hydrolytic activity. ClbS also lacks the metal-binding motif found in other members of its superfamily, harboring instead a tyrosine residue in place of the metal ion. Computational docking of the ClbS substrate into the active site placed the cyclopropane moiety in proximity to this tyrosine residue, and its mutation to phenylalanine abolished the hydrolytic activity of ClbS, implicating tyrosine as a catalytic residue. The identification of ClbS as a cyclopropane hydrolase provides insights into the resistance and toxicity mechanisms of colibactin and could be useful in developing treatment strategies.