Reactivity of an Unusual Amidase May Explain Colibactin’s DNA Cross-Linking Activity

Abstract

Certain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though 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.

ClbR Is the Key Transcriptional Activator of Colibactin Gene Expression in Escherichia coli

“…Furthermore, components of the colibactin biosynthesis machinery are also involved in microcin M and H47 biosynthesis (3). Extensive efforts have been invested into the elucidation of the biosynthesis pathway, structure, and mode of action of this secondary metabolite (11,(18)(19)(20) in order to understand its biological function (21).…”

Two Polyketides Intertwined in Complex Regulation: Posttranscriptional CsrA-Mediated Control of Colibactin and Yersiniabactin Synthesis in Escherichia coli

“…Different colibactin structures have been suggested recently ( 23 – 26 ). Colibactin mediates cross-linking of DNA, in vitro as well as in vivo , resulting in DNA double-strand breaks, thus provoking cell cycle arrest and inducing megalocytosis/senescence ( 22 , 25 – 27 ). This genotoxin promotes bacterial virulence ( 28 , 29 ), but the ability to produce colibactin has also been correlated with probiotic and analgesic effects ( 30 , 31 ), potential antimicrobial activity ( 32 ), and cancer propagation ( 33 – 37 ).…”

Uropathogenic E. coli induces DNA damage in the bladder

“…Colibactin is first synthesised as an inactive prodrug by ClbN followed by the sequential interventions of multiple Clb enzymes. The ClbP peptidase subsequently cleaves the C14-Asparagine (C14-Asn) motif thereby releasing the mature, active form of colibactin with its twin warheads ( Fig 1A ) [ 15 – 17 ]. The genotoxin alkylates adenine residues on both strands of DNA, producing DNA interstrand cross-links [ 18 – 20 ].…”