<i>In Vitro</i> Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

Volpe, Matthew R.; Wilson, Michael; Brotherton, Carolyn A.; Winter, Ethan S.; Johnson, Sheila; Balskus, Emily P.

doi:10.1021/acschembio.9b00069

Cited by 12 publications

(23 citation statements)

References 21 publications

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“…It might, therefore be assumed that the widespread use and overuse of antibiotics, such as β-lactams by humans over the last century has altered this co-evolution, forcing bacteria to develop β-lactamases. The clbP gene, which is part of the colibactin gene cluster (55-Kb), is responsible for the production and expression of the colibactin drug by the E. coli IHE3034 isolate and is an important component of colibactin’s prodrug resistance mechanism [ 28 , 29 , 30 , 31 ]. Furthermore, studies have shown that ClbP is a member of a novel subfamily of extra cytoplasmic serine-reactive peptidases that operate as NRP compound maturation enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, studies have shown that ClbP is a member of a novel subfamily of extra cytoplasmic serine-reactive peptidases that operate as NRP compound maturation enzymes. The critical role of ClbP in the formation of the biological impact makes it a key target for controlling the bioactivity of the PK-NRP-producing pks gene cluster, which may affect commensalism and/or pathogenicity and serve as a risk factor for the development of colorectal cancer [ 15 , 18 , 28 , 31 , 32 ]. In this study, the clbP gene was evaluated in silico and expressed in vitro in order to categorise its β-lactamase activity.…”

Section: Discussionmentioning

confidence: 99%

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clbP Gene, a Potential New Member of the β-Lactamase Family

Azour

Al-Bayssari

Pinault

et al. 2022

IJMS

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The colibactin island (pks) of Escherichia coli formed by 19 genes (55-Kb), encodes non-ribosomal peptide (NRP) and polyketide (PK) synthases, which allow the synthesis of colibactin, a suspected hybrid PK-NRP compound that causes damage to DNA in eukaryotic cells. The clbP, an unusual essential gene, is found in the operon structure with the clbS gene in the pks-encoded machinery. Interestingly, the clbP gene has been annotated as a β-lactamase but no previous study has reported its β-lactamase characteristics. In this study, we (i) investigated the β-lactamase properties of the clbP gene in silico by analysing its phylogenetic relationship with bacterial β-lactamase and peptidase enzymes, (ii) compared its three-dimensional (3D) protein structure with those of bacterial β-lactamase proteins using the Phyr2 database and PyMOL software, and (iii) evaluated in vitro its putative enzymatic activities, including β-lactamase, nuclease, and ribonuclease using protein expression and purification from an E. coli BL21 strain. In this study, we reveal a structural configuration of toxin/antitoxin systems in this island. Thus, similar to the toxin/antitoxin systems, the role of the clbP gene within the pks-island gene group appears as an antitoxin, insofar as it is responsible for the activation of the toxin, which is colibactin. In silico, our analyses revealed that ClbP belonged to the superfamily of β-lactamase, class C. Furthermore, in vitro we were unable to demonstrate its β-lactamase activity, likely due to the fact that the clbP gene requires co-expression with other genes, such as the genes present in the pks-island (19 genes). More research is needed to better understand its actions, particularly with regards to antibiotics, and to discover whether it has any additional functions due to the importance of this gene and its toxicity.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

clbP Gene, a Potential New Member of the β-Lactamase Family

Azour

Al-Bayssari

Pinault

et al. 2022

IJMS

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“…Finally, we tested ClbP's activity toward synthetic substrate analogs and found it selectively hydrolyzes the Nacyl-D-asparagine motif both in vitro and in vivo. 3,41 Together, these results suggested that colibactin is initially synthesized as an inactive precursor (precolibactin) and that removal of the prodrug motif by ClbP generates the active genotoxin. Indeed, genetic analyses showed that the catalytic activity of ClbP is required for genotoxicity.…”

Section: Natural Product Structural Elucidation and Discoverymentioning

confidence: 91%

Leveraging Microbial Genomes and Genomic Context for Chemical Discovery

Kountz

Balskus

2021

Acc. Chem. Res.

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Conspectus The genomic era has dramatically changed how we discover and investigate microbial biochemistry. In particular, the exponential expansion in the number of sequenced microbial genomes provides investigators with a vast wealth of sequence data to exploit for the discovery of biochemical functions and mechanisms, as well as novel enzymes and metabolites. In contrast to early biochemical work, which was largely characterized by “forward” approaches that proceed from biomass to enzyme to gene, the availability of genome sequences enables the discovery of new microbial metabolic activities, enzymes, and metabolites by “reverse” approaches that originate with genetic information or by approaches that incorporate features of both forward and reverse methodologies. In the genomic era, the canonical organization of microbial genomes into gene clusters presents a singular opportunity for the utilization of genomic data. Specifically, genomic context (information gleaned from the genes surrounding a gene of interest in the chromosome) is a powerful tool for chemical discovery in microbial systems because of the functional and/or physiological relationship that usually exists between genes found within a gene cluster. This means that the investigator can use this inferred link to generate hypotheses about the functions of individual genes in the cluster or even the function of the entire cluster itself. Here, we discuss how analysis of genomic context in combination with a mechanistic understanding of enzymes can facilitate numerous facets of microbial biochemical research including the identification of biosynthetic gene clusters, the discovery of important and novel enzymes, the elucidation of natural product structures, and the identification of new metabolic pathways. We highlight work from our laboratory using genomic context to discover and study biosynthetic pathways that produce natural products, including the cylindrocyclophanes, nitrogen–nitrogen bond-containing metabolites, and the gut microbial genotoxin colibactin. Although use of genomic context is most commonly associated with studies of natural product biosynthesis, we also show that it can be applied to the study of primary metabolism. We illustrate this with examples from our work studying the members of the glycyl radical enzyme superfamily involved in choline and 4-hydroxyproline degradation in the human gut. Looking forward, we envision increased opportunities to use such information, with the combination of biochemical knowledge and computational tools poised to fuel a new revolution in our ability to connect genes and their biochemical functions. In particular, we note a need for methods that computationally formalize the functional association between genes when such associations are not obvious from manual gene annotations. Such tools will drastically augment the feasibility and scope of gene cluster analysis and accelerate the discovery of new microbial enzymes, metabolites, and metabolic processes.

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“…ClbP residues S95, K98 and Y186 are indispensable for enzymatic activity, and, in a crystal structure of the periplasmic domain, these residues converge to form the catalytic site 19 . ClbP cleaves precolibactin analogs with varied acyl chains and amide substituents but is highly specific toward the d -asparagine sidechain 20 . In the absence of substrate-bound structures, the mechanism underlying the substrate specificity of ClbP is not yet known.…”

Section: Mainmentioning

confidence: 99%

Structural basis of colibactin activation by the ClbP peptidase

et al. 2022

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Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

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In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe

Cited by 12 publications

References 21 publications

clbP Gene, a Potential New Member of the β-Lactamase Family

clbP Gene, a Potential New Member of the β-Lactamase Family

Leveraging Microbial Genomes and Genomic Context for Chemical Discovery

Structural basis of colibactin activation by the ClbP peptidase

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