Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis

Eric P. Trautman, Alan Healy, Emilee E. Shine, Seth B. Herzon, Jason M. Crawford

Research output: Contribution to journalArticle

Abstract

Modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) comprise giant multidomain enzymes responsible for the “assembly line” biosynthesis of many genetically encoded small molecules. Site-directed mutagenesis, protein biochemical, and structural studies have focused on elucidating the catalytic mechanisms of individual multidomain proteins and protein domains within these megasynthases. However, probing their functions at the cellular level typically has invoked the complete deletion (or overexpression) of multidomain-encoding genes or combinations of genes and comparing those mutants with a control pathway. Here we describe a “domain-targeted” metabolomic strategy that combines genome editing with pathway analysis to probe the functions of individual PKS and NRPS catalytic domains at the cellular metabolic level. We apply the approach to the bacterial colibactin pathway, a genotoxic NRPS-PKS hybrid pathway found in certain Escherichia coli. The pathway produces precolibactins, which are converted to colibactins by a dedicated peptidase, ClbP. Domain-targeted metabolomics enabled the characterization of “multidomain signatures”, or functional readouts of NRPS-PKS domain contributions to the pathway-dependent metabolome. These multidomain signatures provided experimental support for individual domain contributions to colibactin biosynthesis and delineated the assembly line timing events of colibactin heterocycle formation. The analysis also led to the structural characterization of two reactive precolibactin metabolites. We demonstrate the fate of these reactive intermediates in the presence and absence of ClbP, which dictates the formation of distinct product groups resulting from alternative cyclization cascades. In the presence of the peptidase, the reactive intermediates are converted to a known genotoxic scaffold, providing metabolic support of our mechanistic model for colibactin-induced genotoxicity. Domain-targeted metabolomics could be more widely used to characterize NRPS-PKS pathways with unprecedented genetic and metabolic precision.

Original languageEnglish (US)
Pages (from-to)4195-4201
Number of pages7
JournalJournal of the American Chemical Society
Volume139
Issue number11
DOIs
StatePublished - Mar 22 2017

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Peptide Synthases
Polyketide Synthases
Metabolomics
Biosynthesis
Peptides
Proteins
Peptide Hydrolases
Genes
Mutagenesis
Gene encoding
Metabolome
Cyclization
Metabolites
Site-Directed Mutagenesis
Scaffolds
Escherichia coli
Catalytic Domain
Enzymes
colibactin
Molecules

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis. / Trautman, Eric P.; Healy, Alan; Shine, Emilee E.; Herzon, Seth B.; Crawford, Jason M.

In: Journal of the American Chemical Society, Vol. 139, No. 11, 22.03.2017, p. 4195-4201.

Research output: Contribution to journalArticle

Trautman, Eric P. ; Healy, Alan ; Shine, Emilee E. ; Herzon, Seth B. ; Crawford, Jason M. / Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis. In: Journal of the American Chemical Society. 2017 ; Vol. 139, No. 11. pp. 4195-4201.
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