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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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Proposed assembly line biosynthetic model for precolibactin A(a) Proposed biosynthesis for the clb assembly line derailment product 15 and its structurally related shunt metabolites (1, 10-21; Supplementary Table S5). Experimentally supported metabolites are indicated with bold numbers, which can result from thioester hydrolysis. Arrows represent NRPS and PKS enzymes with each acronym representing a distinct catalytic domain. Malonyl-CoA and amino acid substrate incorporations, supported by universally 13C-labeled amino acid feeding experiments, are indicated at their proposed cognate carrier proteins (T domains). (b) Predicted and experimentally supported assembly line biosynthesis of advanced derailment products (24-31) and precolibactin A (32). Our studies indicate that the proposed ClbH-dependent ACC formation is ultimately derived from the aminobutyryl moiety of L-Met. (c) Proposed structure of precolibactin A (32) and detected N-acyl-D-Asn ClbP cleavage products (1-9; Supplementary Table S5). NRPS and PKS domains: C, condensation; A, adenylation; T, thiolation sequence of acyl- and peptidyl-carrier proteins; E, epimerization; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclization; Ox, oxidase; TE, thioesterase. *, Denotes evolutionarily deteriorated cis-AT domain in a trans-AT PKS. The bioinformatics predicted thiazoline and thiazole-containing tail was supported by HRMS, MS/MS, and isotopic labeling studies, and its predicted heterocycle order and stereochemistry (#) need further validation.
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Figure 3: Proposed assembly line biosynthetic model for precolibactin A(a) Proposed biosynthesis for the clb assembly line derailment product 15 and its structurally related shunt metabolites (1, 10-21; Supplementary Table S5). Experimentally supported metabolites are indicated with bold numbers, which can result from thioester hydrolysis. Arrows represent NRPS and PKS enzymes with each acronym representing a distinct catalytic domain. Malonyl-CoA and amino acid substrate incorporations, supported by universally 13C-labeled amino acid feeding experiments, are indicated at their proposed cognate carrier proteins (T domains). (b) Predicted and experimentally supported assembly line biosynthesis of advanced derailment products (24-31) and precolibactin A (32). Our studies indicate that the proposed ClbH-dependent ACC formation is ultimately derived from the aminobutyryl moiety of L-Met. (c) Proposed structure of precolibactin A (32) and detected N-acyl-D-Asn ClbP cleavage products (1-9; Supplementary Table S5). NRPS and PKS domains: C, condensation; A, adenylation; T, thiolation sequence of acyl- and peptidyl-carrier proteins; E, epimerization; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclization; Ox, oxidase; TE, thioesterase. *, Denotes evolutionarily deteriorated cis-AT domain in a trans-AT PKS. The bioinformatics predicted thiazoline and thiazole-containing tail was supported by HRMS, MS/MS, and isotopic labeling studies, and its predicted heterocycle order and stereochemistry (#) need further validation.

Mentions: Merging our new understanding of the spirobicyclic structure with retrospective bioinformatic analyses of the biosynthetic pathway and metabolomic network analyses, we predicted the structures for the two other targeted advanced precolibactins (m/z 609.3862 and m/z 816.3780). Our proposed structures were supported by HRMS and MS/MS fragmentation (Supplementary Figs. S18-S20). To gain additional structural support for all detectable clb pathway-dependent metabolites, including these highly unstable advanced precolibactins, we conducted an extensive series of isotopic labeling studies using universally [U-13C]-labeled L-amino acids in an E. coli BW25113 parent strain and in a variety of amino acid auxotrophic strain variants (Supplementary Table S1). Studies were conducted using a control bacterial artificial chromosome (clb-) and the IHE3034-derived bacterial artificial chromosomes bearing clb+ and ΔclbP colibactin pathways. Labeling studies in defined minimal media were guided by bioinformatic predictions of adenylation (A)-domain specificities (Supplementary Fig. S1), established NRPS-PKS logic, and our structural characterizations of the two shunt precolibactins 1517 and 27. By comparing HRMS analyses of 13C-labeled cultures to our detectable comprehensive list of clb-dependent metabolites, we determined system-wide specific L-amino acid (Asn, Ala, Met, Gly, Cys, Ser) isotopic incorporations (Fig. 2, Supplementary Table S2). From this data, 32 predicted metabolites (Supplementary Table S5) were mapped onto the biosynthetic pathway model (Fig. 3). The labeling studies below are described in the context of this model.


The colibactin warhead crosslinks DNA.

Vizcaino MI, Crawford JM - Nat Chem (2015)

Proposed assembly line biosynthetic model for precolibactin A(a) Proposed biosynthesis for the clb assembly line derailment product 15 and its structurally related shunt metabolites (1, 10-21; Supplementary Table S5). Experimentally supported metabolites are indicated with bold numbers, which can result from thioester hydrolysis. Arrows represent NRPS and PKS enzymes with each acronym representing a distinct catalytic domain. Malonyl-CoA and amino acid substrate incorporations, supported by universally 13C-labeled amino acid feeding experiments, are indicated at their proposed cognate carrier proteins (T domains). (b) Predicted and experimentally supported assembly line biosynthesis of advanced derailment products (24-31) and precolibactin A (32). Our studies indicate that the proposed ClbH-dependent ACC formation is ultimately derived from the aminobutyryl moiety of L-Met. (c) Proposed structure of precolibactin A (32) and detected N-acyl-D-Asn ClbP cleavage products (1-9; Supplementary Table S5). NRPS and PKS domains: C, condensation; A, adenylation; T, thiolation sequence of acyl- and peptidyl-carrier proteins; E, epimerization; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclization; Ox, oxidase; TE, thioesterase. *, Denotes evolutionarily deteriorated cis-AT domain in a trans-AT PKS. The bioinformatics predicted thiazoline and thiazole-containing tail was supported by HRMS, MS/MS, and isotopic labeling studies, and its predicted heterocycle order and stereochemistry (#) need further validation.
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Related In: Results  -  Collection

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Figure 3: Proposed assembly line biosynthetic model for precolibactin A(a) Proposed biosynthesis for the clb assembly line derailment product 15 and its structurally related shunt metabolites (1, 10-21; Supplementary Table S5). Experimentally supported metabolites are indicated with bold numbers, which can result from thioester hydrolysis. Arrows represent NRPS and PKS enzymes with each acronym representing a distinct catalytic domain. Malonyl-CoA and amino acid substrate incorporations, supported by universally 13C-labeled amino acid feeding experiments, are indicated at their proposed cognate carrier proteins (T domains). (b) Predicted and experimentally supported assembly line biosynthesis of advanced derailment products (24-31) and precolibactin A (32). Our studies indicate that the proposed ClbH-dependent ACC formation is ultimately derived from the aminobutyryl moiety of L-Met. (c) Proposed structure of precolibactin A (32) and detected N-acyl-D-Asn ClbP cleavage products (1-9; Supplementary Table S5). NRPS and PKS domains: C, condensation; A, adenylation; T, thiolation sequence of acyl- and peptidyl-carrier proteins; E, epimerization; KS, ketosynthase; AT, acyl-transferase; KR, ketoreductase; DH, dehydratase; ER, enoyl-reductase; Cy, condensation/cyclization; Ox, oxidase; TE, thioesterase. *, Denotes evolutionarily deteriorated cis-AT domain in a trans-AT PKS. The bioinformatics predicted thiazoline and thiazole-containing tail was supported by HRMS, MS/MS, and isotopic labeling studies, and its predicted heterocycle order and stereochemistry (#) need further validation.
Mentions: Merging our new understanding of the spirobicyclic structure with retrospective bioinformatic analyses of the biosynthetic pathway and metabolomic network analyses, we predicted the structures for the two other targeted advanced precolibactins (m/z 609.3862 and m/z 816.3780). Our proposed structures were supported by HRMS and MS/MS fragmentation (Supplementary Figs. S18-S20). To gain additional structural support for all detectable clb pathway-dependent metabolites, including these highly unstable advanced precolibactins, we conducted an extensive series of isotopic labeling studies using universally [U-13C]-labeled L-amino acids in an E. coli BW25113 parent strain and in a variety of amino acid auxotrophic strain variants (Supplementary Table S1). Studies were conducted using a control bacterial artificial chromosome (clb-) and the IHE3034-derived bacterial artificial chromosomes bearing clb+ and ΔclbP colibactin pathways. Labeling studies in defined minimal media were guided by bioinformatic predictions of adenylation (A)-domain specificities (Supplementary Fig. S1), established NRPS-PKS logic, and our structural characterizations of the two shunt precolibactins 1517 and 27. By comparing HRMS analyses of 13C-labeled cultures to our detectable comprehensive list of clb-dependent metabolites, we determined system-wide specific L-amino acid (Asn, Ala, Met, Gly, Cys, Ser) isotopic incorporations (Fig. 2, Supplementary Table S2). From this data, 32 predicted metabolites (Supplementary Table S5) were mapped onto the biosynthetic pathway model (Fig. 3). The labeling studies below are described in the context of this model.

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