Certain commensal and pathogenic bacteria produce colibactin, a small-molecule genotoxin that causes interstrand cross-links in host cell DNA. Although colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and β-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line and incorporating two electrophilic cyclopropane warheads into the final natural product scaffold. This proposed transformation was supported by the detection of a colibactin-derived cross-linked DNA adduct. Overall, this work provides a biosynthetic explanation for colibactin's DNA cross-linking activity and paves the way for further study of its chemical structure and biological roles.
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We thank Alex Sieg for help with cloning, Lihan Zhang for help with NMR experiments, and Matthew Volpe for comments on the manuscript. We acknowledge financial support from the National Institutes of Health (R01CA208834-02) (E.P.B.). Salary support for P.W.V. was provided by the U.S. National Institutes of Health and National Cancer Institute [Grant R50-CA211256]. Mass spectrometry for DNA adduct analysis was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, supported in part by the U.S. National Institutes of Health and National Cancer Institute [Cancer Center Support Grant CA-77598]. M.R.W. acknowledges support from the American Cancer Society-New England Division Postdoctoral Fellowship PF-16-122-01-CDD.