Advancing the Biosynthetic and Chemical Understanding of the Carcinogenic Risk Factor Colibactin and Its Producers

Hirayama, Yoshitaka; Sato, Michio; Watanabe, Kenji

doi:10.1021/acs.biochem.2c00229

Cited by 4 publications

(1 citation statement)

References 51 publications

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“…N-Deacylation of precolibactins by the periplasmic peptidase ClbP leads to the formation of the active genotoxin colibactin (Velilla et al, 2023;Volpe et al, 2023). Isolation and structure determination of colibactin has been a challenge because of product instability; however, several precolibactins have been isolated and the bioactive product has been characterized (Williams et al, 2020;Tang et al, 2022;Wernke et al, 2020;Hirayama et al, 2022). Complementary to this, clb+ Escherichia coli have been shown to generate DNA interstrand crosslinks, both in vivo and in vitro, via N7 adenine alkylation by the electrophilic cyclopropane ring of colibactin (Wilson et al, 2019;Xue et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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

Tripathi

Mousa

Guntaka

et al. 2023

Acta Crystallogr D Struct Biol

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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.

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Section: Introductionmentioning

confidence: 99%

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

Tripathi

Mousa

Guntaka

et al. 2023

Acta Crystallogr D Struct Biol

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The Rapid Synthesis of Colibactin Warhead Model Compounds Using New Metal‐Free Photocatalytic Cyclopropanation Reactions Facilitates the Investigation of Biological Mechanisms

Bosveli

Griboura

Kampouropoulos

et al. 2023

Chemistry A European J

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Herein, we report the synthesis of a series of colibactin warhead model compounds using two newly developed metal‐free photocatalytic cyclopropanation reactions. These mild cyclopropanations expand the known applications of eosin within synthesis. A halogen atom transfer reaction mode has been harnessed so that dihalides can be used as the cyclopropanating agents. The colibactin warhead models were then used to provide new insight into two key mechanisms in colibactin chemistry. An explanation is provided for why the colibactin warhead sometimes undergoes a ring expansion‐addition reaction to give fused cyclobutyl products while at other times nucleophiles add directly to the cyclopropyl unit (as when DNA adds to colibactin). Finally, we provide some evidence that Cu(II) chelated to colibactin may catalyze an important oxidation of the colibactin‐DNA adduct. The Cu(I) generated as a result could then also play a role in inducing double strand breaks in DNA.

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Structure Elucidation of Secondary Metabolites: Current Frontiers and Lingering Pitfalls

et al. 2023

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Conspectus Analytical methods allow for the structure determination of submilligram quantities of complex secondary metabolites. This has been driven in large part by advances in NMR spectroscopic capabilities, including access to high-field magnets equipped with cryogenic probes. Experimental NMR spectroscopy may now be complemented by remarkably accurate carbon-13 NMR calculations using state-of-the-art DFT software packages. Additionally, microED analysis stands to have a profound effect on structure elucidation by providing X-ray-like images of microcrystalline samples of analytes. Nonetheless, lingering pitfalls in structure elucidation remain, particularly for isolates that are unstable or highly oxidized. In this Account, we discuss three projects from our laboratory that highlight nonoverlapping challenges to the field, with implications for chemical, synthetic, and mechanism of action studies. We first discuss the lomaiviticins, complex unsaturated polyketide natural products disclosed in 2001. The original structures were derived from NMR, HRMS, UV–vis, and IR analysis. Owing to the synthetic challenges presented by their structures and the absence of X-ray crystallographic data, the structure assignments remained untested for nearly two decades. In 2021, the Nelson group at Caltech carried out microED analysis of (−)-lomaiviticin C, leading to the startling discovery that the original structure assignment of the lomaiviticins was incorrect. Acquisition of higher-field (800 MHz 1H, cold probe) NMR data as well as DFT calculations provided insights into the basis for the original misassignment and lent further support to the new structure identified by microED. Reanalysis of the 2001 data set reveals that the two structure assignments are nearly indistinguishable, underscoring the limitations of NMR-based characterization. We then discuss the structure elucidation of colibactin, a complex, nonisolable microbiome metabolite implicated in colorectal cancer. The colibactin biosynthetic gene cluster was detected in 2006, but owing to colibactin’s instability and low levels of production, it could not be isolated or characterized. We used a combination of chemical synthesis, mechanism of action studies, and biosynthetic analysis to identify the substructures in colibactin. These studies, coupled with isotope labeling and tandem MS analysis of colibactin-derived DNA interstrand cross-links, ultimately led to a structure assignment for the metabolite. We then discuss the ocimicides, plant secondary metabolites that were studied as agents against drug-resistant P. falciparum. We synthesized the core structure of the ocimicides and found significant discrepancies between our experimental NMR spectroscopic data and that reported for the natural products. We determined the theoretical carbon-13 NMR shifts for 32 diastereomers of the ocimicides. These studies indicated that a revision of the connectivity of the metabolites is likely needed. We end with some thoughts on the frontiers of secondary metabolite structure...

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Advancing the Biosynthetic and Chemical Understanding of the Carcinogenic Risk Factor Colibactin and Its Producers

Cited by 4 publications

References 51 publications

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

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

The Rapid Synthesis of Colibactin Warhead Model Compounds Using New Metal‐Free Photocatalytic Cyclopropanation Reactions Facilitates the Investigation of Biological Mechanisms

Structure Elucidation of Secondary Metabolites: Current Frontiers and Lingering Pitfalls

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