title: Domain-Targeted Metabolomics Delineates the Heterocycle Assembly Steps of Colibactin Biosynthesis.
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.
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.
authors.forename: Eric P
authors.identifier: 0000-0002-0974-2356
authors.initials: EP
authors.lastname: Trautman
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Healy
authors.affiliation: Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.|Department of Microbial Pathogenesis, Yale School of Medicine , New Haven, Connecticut 06536, United States.
authors.forename: Emilee E
authors.initials: EE
authors.lastname: Shine
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Department of Pharmacology, Yale School of Medicine , New Haven, Connecticut 06520, United States.
authors.forename: Seth B
authors.identifier: 0000-0001-5940-9853
authors.initials: SB
authors.lastname: Herzon
authors.affiliation: Department of Chemistry, Yale University , New Haven, Connecticut 06520, United States.|Chemical Biology Institute, Yale University , West Haven, Connecticut 06516, United States.|Department of Microbial Pathogenesis, Yale School of Medicine , New Haven, Connecticut 06536, United States.
authors.forename: Jason M
authors.identifier: 0000-0002-7583-1242
authors.initials: JM
authors.lastname: Crawford
mesh_terms: D004926:Escherichia coli / Q000201:enzymology; D006571:Heterocyclic Compounds / Q000737:chemistry / Q000378:metabolism*; D055432:Metabolomics; D008968:Molecular Conformation; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006571:Heterocyclic Compounds; D010455:Peptides; D061065:Polyketides; C569566:colibactin; D048630:Polyketide Synthases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase