ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance

Abstract

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

Structure elucidation of colibactin

“…S81-S88 and Tables S10, S14, S18, S22, S26, S30, S35, S39, S43). The aminal functional groups in 9 derive from aerobic oxidation 5 of the ring-opened products, as previously established (25,38).…”

“…Deletion of the clb genomic island (16) in Nissle 1917 destroyed production, as expected. To improve titers for robust isotopic labeling, we constructed a clbS colibactin resistance (38,40) mutant in Nissle 1917. While deletion of clbS leads to a 5 fitness defect and activates a clb-dependent bacterial SOS DNA damage response (40), this genetic modification resulted in an 8.5-fold improvement in the mass spectral signal intensity of colibactin (17).…”

“…While deletion of clbS leads to a 5 fitness defect and activates a clb-dependent bacterial SOS DNA damage response (40), this genetic modification resulted in an 8.5-fold improvement in the mass spectral signal intensity of colibactin (17). This is likely due to decreased processing of colibactin (17) by ClbS-mediated hydrolytic opening of the cyclopropane (38).…”

Photoactivated Colibactin Probes Induce Cellular DNA Damage

“…[4] Furthermore, pks + E. coli enhance in vivo tumour growth in mice that are predisposed to CRC. It has been proposed that this prodrug mechanism, in combination with the production of the protective protein ClbS, [8] reduces autotoxicity.G iven these factors, colibactin has been the subject of several reviews. After export into the periplasmic space, [6] the precolibactin is cleaved by the peptidase ClbP to yield the benign Nmyristoyl-d-asparagine [7] and the active genotoxic colibactin "warhead".…”

“…After export into the periplasmic space, [6] the precolibactin is cleaved by the peptidase ClbP to yield the benign Nmyristoyl-d-asparagine [7] and the active genotoxic colibactin "warhead". It has been proposed that this prodrug mechanism, in combination with the production of the protective protein ClbS, [8] reduces autotoxicity.G iven these factors, colibactin has been the subject of several reviews. [9] To date,the isolation of acolibactin metabolite produced by the full complement of biosynthetic genes required for in vivo genotoxicity has proven elusive.F urthermore,n o deacylated active colibactins have been isolated, presumably due to low production and instability.…”