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Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin.

ABSTRACT: Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS-PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL couples α-aminoketone and β-ketothioester intermediates attached to separate carrier domains on the NRPS-PKS assembly. Here, the 1.9 Å resolution structure of ClbL is reported, providing a structural basis for this key step in the colibactin biosynthetic pathway. The structure reveals an open hydrophobic active site surrounded by flexible loops, and comparison with homologous amidases supports its unusual function and predicts macromolecular interactions with pathway carrier-protein substrates. Modeling protein-protein interactions supports a predicted molecular basis for enzyme-carrier domain interactions. Overall, the work provides structural insight into this unique enzyme that is central to the biosynthesis of colibactin.

SUBMITTER: Tripathi P

PROVIDER: S-EPMC10478638 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin.

Tripathi Prabhanshu P Mousa Jarrod J JJ Guntaka Naga Sandhya NS Bruner Steven D SD

Acta crystallographica. Section D, Structural biology 20230810 Pt 9

Colibactin is a genotoxic natural product produced by select commensal bacteria in the human gut microbiota. The compound is a bis-electrophile that is predicted to form interstrand DNA cross-links in target cells, leading to double-strand DNA breaks. The biosynthesis of colibactin is carried out by a mixed NRPS-PKS assembly line with several noncanonical features. An amidase, ClbL, plays a key role in the pathway, catalyzing the final step in the formation of the pseudodimeric scaffold. ClbL co ...[more]

PMID: 37561403

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Project description:The genotoxin colibactin produced by resident bacteria of the gut microbiota may have tumorigenic effect by inducing DNA double-strand breaks in host cells. Yet, the effect of colibactin on gut microbiota composition and functions remains unknown. To address this point, we designed an experiment in which pregnant mice were colonized with the following: (i) a commensal Escherichia coli strain, (ii) a commensal E. coli strain plus a genotoxic E. coli strain, (iii) a commensal E. coli strain plus a nongenotoxic E. coli mutant strain unable to produce mature colibactin. Then, we analyzed the gut microbiota in pups at day 15 and day 35 after birth. At day 15, mice that were colonized at birth with the genotoxic strain showed lower levels of Proteobacteria and taxa belonging to the Proteobacteria, a modest effect on overall microbial diversity, and no effect on gut microbiome. At day 35, mice that received the genotoxic strain showed lower Firmicutes and taxa belonging to the Firmicutes, together with a strong effect on overall microbial diversity and higher microbial functions related to DNA repair. Moreover, the genotoxic strain strongly affected gut microbial diversity evolution of pups receiving the genotoxic strain between day 15 and day 35. Our data show that colibactin, beyond targeting the host, may also exert its genotoxic effect on the gut microbiota.IMPORTANCE Infections of genotoxic Escherichia coli spread concomitantly with urbanized progression. These bacteria may prompt cell senescence and affect DNA stability, inducing cancer via the production of colibactin, a genotoxin shown capable of affecting host DNA in eukaryotic cells. In this study, we show that the action of colibactin may also be directed against other bacteria of the gut microbiota in which genotoxic E. coli bacteria have been introduced. Indeed, the presence of genotoxic E. coli induced a change in both the structure and function of the gut microbiota. Our data indicate that genotoxic E. coli may use colibactin to compete for gut niche utilization.

Project description:Colibactins are hybrid polyketide-nonribosomal peptides produced by Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae harboring the pks genomic island. These genotoxic metabolites are produced by pks-encoded peptide-polyketide synthases as inactive prodrugs called precolibactins, which are then converted to colibactins by deacylation for DNA-damaging effects. Colibactins are bona fide virulence factors and are suspected of promoting colorectal carcinogenesis when produced by intestinal E. coli Natural active colibactins have not been isolated, and how they induce DNA damage in the eukaryotic host cell is poorly characterized. Here, we show that DNA strands are cross-linked covalently when exposed to enterobacteria producing colibactins. DNA cross-linking is abrogated in a clbP mutant unable to deacetylate precolibactins or by adding the colibactin self-resistance protein ClbS, confirming the involvement of the mature forms of colibactins. A similar DNA-damaging mechanism is observed in cellulo, where interstrand cross-links are detected in the genomic DNA of cultured human cells exposed to colibactin-producing bacteria. The intoxicated cells exhibit replication stress, activation of ataxia-telangiectasia and Rad3-related kinase (ATR), and recruitment of the DNA cross-link repair Fanconi anemia protein D2 (FANCD2) protein. In contrast, inhibition of ATR or knockdown of FANCD2 reduces the survival of cells exposed to colibactin-producing bacteria. These findings demonstrate that DNA interstrand cross-linking is the critical mechanism of colibactin-induced DNA damage in infected cells.IMPORTANCE Colorectal cancer is the third-most-common cause of cancer death. In addition to known risk factors such as high-fat diets and alcohol consumption, genotoxic intestinal Escherichia coli bacteria producing colibactin are proposed to play a role in colon cancer development. Here, by using transient infections with genotoxic E. coli, we showed that colibactins directly generate DNA cross-links in cellulo Such lesions are converted into double-strand breaks during the repair response. DNA cross-links, akin to those induced by metabolites of alcohol and high-fat diets and by widely used anticancer drugs, are both severely mutagenic and profoundly cytotoxic lesions. This finding of a direct induction of DNA cross-links by a bacterium should facilitate delineating the role of E. coli in colon cancer and engineering new anticancer agents.

Dataset Information

Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin.

Publications

Structural basis of the amidase ClbL central to the biosynthesis of the genotoxin colibactin.

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