Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin

Li, Zhong Rui; Li, Jie; Gu, Jin Ping; Lai, Jennifer Y. H.; Duggan, Brendan M.; Zhang, Weipeng; Li, Zhi Long; Li, Yongxin; Tong, Rongbiao; Xü, Ying; Lin, Dongguo; Moore, Bradley S.; Qian, Pei‐Yuan

doi:10.1038/nchembio.2157

Cited by 76 publications

(154 citation statements)

References 33 publications

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“…In plants, ACC is an intermediate in ethylene biosynthesis and is derived from methionine via a pyridoxal phosphate (PLP)-dependent mechanism. 14 ACC is not known to be produced by E. coli, and while isotopic labeling studies have shown that the ACC moiety in precolibactins is derived from methionine, 11b,d the colibactin gene cluster does not encode apparent PLP-dependent enzymes. ClbH, the bio-synthetic enzyme that incorporates this moiety, has an unusual domain architecture for an NRPS.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to the canonical C–A–T module (containing condensation, adenylation, and thiolation domains), ClbH contains a second N-terminal A domain, A 1 –C–A 2 –T (Figure 2a). Bioinformatic analysis 11b and biochemical studies 11f,15 indicate that the noncanonical A 1 domain activates serine and loads the carrier protein ClbE in trans. Dehydrogenases ClbD and ClbF then oxidize the enzyme-bound serine to produce the polyketide extender unit α -aminomalonyl-ClbE.…”

Section: Resultsmentioning

confidence: 99%

“…Dehydrogenases ClbD and ClbF then oxidize the enzyme-bound serine to produce the polyketide extender unit α -aminomalonyl-ClbE. 11f,15 ClbG transfers the α -aminomalonyl unit onto the T domain of ClbK. 11f It is currently unknown whether additional α -aminomalonyl units are incorporated or compete with malonyl building blocks, a phenomenon that has been observed in vivo for polyketide structural diversification 16 and in in vitro protein biochemical studies.…”

Section: Resultsmentioning

confidence: 99%

“…12 Colibactins are synthesized as prodrug scaffolds called “precolibactins”, which are cleaved in the bacterial periplasm to liberate an N -acyl-D-Asn side chain and genotoxic colibactins. 8a,11a,13 With the available structural information, coarse full gene deletion studies, and select protein biochemical studies, a steadily growing colibactin biosynthetic model has been supported for these observed metabolites. A current biosynthetic model, including the interpretation of new results from this study, is shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

“…The advantage of MAGE is that it enables targeted mutations with single-base-pair resolution. We measured the production of previously reported clb -dependent ions, 8a,11b known (pre)-colibactin metabolites, and some additional biosynthetic products. We report the domain dependencies of each of these metabolites (multidomain signatures, Tables S3–S5).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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Photoactivated Colibactin Probes Induce Cellular DNA Damage

et al. 2018

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Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks+ bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative “warhead” and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks+ like phenotype in HeLa cells. Furthermore, results from DNA cross‐linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

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Nature's Combinatorial Biosynthesis Produces Vatiamides A–F

et al. 2019

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Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide‐based click probe led to the discovery and characterization of vatiamides A–F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14‐2. These derive from a unique combinatorial non‐collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS‐NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.

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Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin

Cited by 76 publications

References 33 publications

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

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

Photoactivated Colibactin Probes Induce Cellular DNA Damage

Nature's Combinatorial Biosynthesis Produces Vatiamides A–F

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