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The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

Bottom Line: Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure.The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity.The data support unexpected models for both colibactin biosynthesis and its mode of action.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

ABSTRACT
Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes 'colibactin', which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

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Key colibactin pathway (clb)-dependent shunt metabolitesAnalogous to the decarboxylation of clb-assembly line derailment product 14 to 15, characterized shunt precolibactin 27 most likely arises from decarboxylation of transient derailment product 26. ClbP-mediated cleavage appears to be promiscuous in our analysis, leading to N-terminal N-acyl-D-asparagines, such as 1, and detectable C-terminal products, such as 21 and 30. Structures are numbered in accordance with increasing biosynthetic complexity as illustrated in Figure 3.
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Figure 1: Key colibactin pathway (clb)-dependent shunt metabolitesAnalogous to the decarboxylation of clb-assembly line derailment product 14 to 15, characterized shunt precolibactin 27 most likely arises from decarboxylation of transient derailment product 26. ClbP-mediated cleavage appears to be promiscuous in our analysis, leading to N-terminal N-acyl-D-asparagines, such as 1, and detectable C-terminal products, such as 21 and 30. Structures are numbered in accordance with increasing biosynthetic complexity as illustrated in Figure 3.

Mentions: Detailed mode-of-action studies for the clb pathway have remained experimentally intractable without its corresponding small molecule structures. However, colibactin’s instability has thwarted structural characterization efforts despite attempts from various labs over the last nine years6. Employing molecular networking tools16, we recently developed a pathway-targeted structural network analysis approach to map the clb pathway within E. coli’s complex metabolome. To validate the approach, we isolated, structurally characterized, and synthesized the most abundant molecular features in the network map, including the first authentic precolibactin assembly line derailment product 1517 (Fig. 1; Structures 1-32 are numbered in order of biosynthetic complexity as detailed below). 15 is proteolytically cleaved by peptidase ClbP to liberate small alkyl-amine 16, which accumulated in organic extracts as cyclic imine 17, and N-myristoyl-D-Asn 1, which exhibited in vitro bacterial growth inhibitory activity and served as an antagonist of the 5-hydroxytryptamine-7 receptor implicated in colitis17. Here we elucidate the structure of the relatively stable precolibactin derailment product 27, illuminating the colibactin warhead, an unprecedented substituted spirobicyclic structural feature. Correspondingly, we employ [U-13C]-isotopic labeling studies in various auxotrophic strain backgrounds in conjunction with pathway-targeted molecular network analyses among wildtype, peptidase clbP mutant, and control pathways to provide a system-wide analysis of colibactin biosynthesis. The data collectively support the structures of highly unstable advanced precolibactins and an unexpected biosynthetic model that accounts for 32 predicted clb-dependent molecular features. In contrast to the reported DNA double-strand break phenotype, we demonstrate that the colibactin warhead crosslinks DNA in vitro, supporting a new model for colibactin’s mode of action.


The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

Key colibactin pathway (clb)-dependent shunt metabolitesAnalogous to the decarboxylation of clb-assembly line derailment product 14 to 15, characterized shunt precolibactin 27 most likely arises from decarboxylation of transient derailment product 26. ClbP-mediated cleavage appears to be promiscuous in our analysis, leading to N-terminal N-acyl-D-asparagines, such as 1, and detectable C-terminal products, such as 21 and 30. Structures are numbered in accordance with increasing biosynthetic complexity as illustrated in Figure 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499846&req=5

Figure 1: Key colibactin pathway (clb)-dependent shunt metabolitesAnalogous to the decarboxylation of clb-assembly line derailment product 14 to 15, characterized shunt precolibactin 27 most likely arises from decarboxylation of transient derailment product 26. ClbP-mediated cleavage appears to be promiscuous in our analysis, leading to N-terminal N-acyl-D-asparagines, such as 1, and detectable C-terminal products, such as 21 and 30. Structures are numbered in accordance with increasing biosynthetic complexity as illustrated in Figure 3.
Mentions: Detailed mode-of-action studies for the clb pathway have remained experimentally intractable without its corresponding small molecule structures. However, colibactin’s instability has thwarted structural characterization efforts despite attempts from various labs over the last nine years6. Employing molecular networking tools16, we recently developed a pathway-targeted structural network analysis approach to map the clb pathway within E. coli’s complex metabolome. To validate the approach, we isolated, structurally characterized, and synthesized the most abundant molecular features in the network map, including the first authentic precolibactin assembly line derailment product 1517 (Fig. 1; Structures 1-32 are numbered in order of biosynthetic complexity as detailed below). 15 is proteolytically cleaved by peptidase ClbP to liberate small alkyl-amine 16, which accumulated in organic extracts as cyclic imine 17, and N-myristoyl-D-Asn 1, which exhibited in vitro bacterial growth inhibitory activity and served as an antagonist of the 5-hydroxytryptamine-7 receptor implicated in colitis17. Here we elucidate the structure of the relatively stable precolibactin derailment product 27, illuminating the colibactin warhead, an unprecedented substituted spirobicyclic structural feature. Correspondingly, we employ [U-13C]-isotopic labeling studies in various auxotrophic strain backgrounds in conjunction with pathway-targeted molecular network analyses among wildtype, peptidase clbP mutant, and control pathways to provide a system-wide analysis of colibactin biosynthesis. The data collectively support the structures of highly unstable advanced precolibactins and an unexpected biosynthetic model that accounts for 32 predicted clb-dependent molecular features. In contrast to the reported DNA double-strand break phenotype, we demonstrate that the colibactin warhead crosslinks DNA in vitro, supporting a new model for colibactin’s mode of action.

Bottom Line: Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure.The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity.The data support unexpected models for both colibactin biosynthesis and its mode of action.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry, Yale University, New Haven, Connecticut 06510, USA [2] Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, USA.

ABSTRACT
Members of the human microbiota are increasingly being correlated to human health and disease states, but the majority of the underlying microbial metabolites that regulate host-microbe interactions remain largely unexplored. Select strains of Escherichia coli present in the human colon have been linked to the initiation of inflammation-induced colorectal cancer through an unknown small-molecule-mediated process. The responsible non-ribosomal peptide-polyketide hybrid pathway encodes 'colibactin', which belongs to a largely uncharacterized family of small molecules. Genotoxic small molecules from this pathway that are capable of initiating cancer formation have remained elusive due to their high instability. Guided by metabolomic analyses, here we employ a combination of NMR spectroscopy and bioinformatics-guided isotopic labelling studies to characterize the colibactin warhead, an unprecedented substituted spirobicyclic structure. The warhead crosslinks duplex DNA in vitro, providing direct experimental evidence for colibactin's DNA-damaging activity. The data support unexpected models for both colibactin biosynthesis and its mode of action.

Show MeSH
Related in: MedlinePlus