The identification of bacillaene, the product of the PksX megacomplex in
            <i>Bacillus subtilis</i>

Butcher, Rebecca A.; Schroeder, Frank C.; Fischbach, Michael A.; Straight, Paul D.; Kolter, Roberto; Walsh, Christopher T.; Clardy, Jon

doi:10.1073/pnas.0610503104

Cited by 247 publications

(241 citation statements)

References 19 publications

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“…Previous experience with identification of novel secondary products in unfractionated microbial extracts suggested that DANS, a method based on overlaying 2D-NMR spectra, could be used to directly detect the daf-22-dependent compounds (17,18). DANS, as shown previously, can provide detailed structural information even for minor components in complex metabolite extracts.…”

Section: Nmr-spectroscopic Comparison Of Wildmentioning

confidence: 96%

“…However, the armamentarium of traditional natural product chemistry appears ill-suited for this purpose, given the complexity of metabolomes and the scope of assigning functions to hundreds if not thousands of individual components, many of which represent previously undescribed chemical structures (15). We have developed an NMR-based approach that simplifies linking naturally occurring small molecules with their biological function and offers considerable advantages for the detection of synergism as well as for the characterization of chemically labile signaling molecules (16)(17)(18). Central to our method is the use of genetic information to detect individual compounds in the C. elegans metabolome that could be of interest within a specific biological context.…”

Section: T He Importance Of Small-molecule Signaling In the Biology Ofmentioning

confidence: 99%

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A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Pungaliya

Srinivasan

Fox

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type C. elegans and a signaling-deficient mutant, daf-22, enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a p-aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure-activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the maleattracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying smallmolecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits.dauer formation ͉ differential analysis ͉ metabolomics ͉ NMR spectroscopy ͉ sex pheromone

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Section: Nmr-spectroscopic Comparison Of Wildmentioning

confidence: 96%

Section: T He Importance Of Small-molecule Signaling In the Biology Ofmentioning

confidence: 99%

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Pungaliya

Srinivasan

Fox

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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357

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“…A recent report has assigned the pks gene cluster to production of bacillaene based on the analysis of an orthologous gene cluster from Bacillus amyloliquefaciens FZB42 called bae (6). In concurrent work, we have solved the structure of bacillaene from B. subtilis and proposed a model for its biosynthesis (28). Bioinformatic analyses of the bacillaene synthase gene cluster reveal features in common with unconventional PKSs from streptomycetes, myxobacteria, and cyanobacteria (see Fig.…”

mentioning

confidence: 95%

A singular enzymatic megacomplex from Bacillus subtilis

Straight

Fischbach²,

Walsh³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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175

168

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Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (Ϸ2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies Ϸ2% of the Bacillus subtilis genome, encodes the subunits of Ϸ2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membraneassociated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs. bacillaene ͉ nonribosomal peptide ͉ polyketide ͉ Streptomyces N onribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes have received much attention in recent years, in large part because of their potential to be engineered to direct the synthesis of novel compounds with therapeutic value. This engineering potential stems from the fact that the individual modules of these enzymes are organized into assembly lines in which the order of modules determines the identity of the polyketide or polypeptide product (1, 2). Each domain in the assembly line specifies the incorporation of an individual substrate, because the nascent peptide or polyketide is passed sequentially between domains. The structures of many individual NRPS and PKS domains have been solved (3), and the structures of mammalian and fungal isoforms of the PKS-like fatty acid synthases were recently reported (4, 5), bringing into focus a picture of individual enzymatic assembly line architecture. However, much of our current knowledge of NRPSs and PKSs comes from studies in which these proteins are expressed individually in heterologous hosts, leaving much to learn about the quaternary organization and cell biology of these massive enzymes in the context of their native producer organisms.The genome of Bacillus subtilis contains the pks gene cluster, which encodes a hybrid NRPS/PKS (Figs. 1A and 4) that was considered nonfunctional until recently. A recent report has assigned the pks gene cluster to production of bacillaene based on the analysis of an orthologous gene cluster from Bacillus amyloliquefaciens FZB42 called bae (6). In concurrent work, we have solved the structure of bacillaene from B. subtilis and proposed a model for its biosynthesis (28). Bioinformatic analyses of the bacillaene synthase gene cluster reveal features in common with unconventional PKSs from streptomycetes, myxobacteria, and cyanobacteria (see Fig. 4). Examples of these features include three trans-acting acyltransferase (AT) domains that introduce substrates to the assembly line (7) and a six-protein subcluster th...

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mentioning

confidence: 99%

“…The hybrid PK/ NRPs dihydrobacillaene and bacillaene are biosignaling agents from Bacillus subtilis where each can be described as a diamide of -amino polyenoic acids that is N-capped with an ␣-hydroxyisocaproate (␣-HIC) moiety (3). The metabolite initially arising from their shared assembly line is dihydrobacillaene, which is later converted to bacillaene by the cytochrome P450 PksS, which oxidizes the saturated C14Ј-C15Ј bond of dihydrobacillaene to an olefin (3)(4)(5). Previous work has shown the dihydrobacillaene assembly line to be a rich source of unusual biochemistry, where hybrid PK synthase (PKS)/NRP synthetase assembly-line proteins are used in conjunction with enzyme machinery borrowed from both isoprenoid and polyunsaturated fatty acid biosynthesis (6).…”

mentioning

confidence: 99%

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

Calderone

Bumpus

Kelleher

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis

Cited by 247 publications

References 19 publications

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

A singular enzymatic megacomplex from Bacillus subtilis

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth

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