Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation.
We have identified a new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine-specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.
SummaryXenocoumacin 1 (Xcn1) and xenocoumacin 2 (Xcn2) are the major antimicrobial compounds produced by Xenorhabdus nematophila. To study the role of Xcn1 and Xcn2 in the life cycle of X. nematophila the 14 gene cluster (xcnA-N) required for their synthesis was identified. Overlap RT-PCR analysis identified six major xcn transcripts. Individual inactivation of the non-ribosomal peptide synthetase genes, xcnA and xcnK, and polyketide synthetase genes, xcnF, xcnH and xcnL, eliminated Xcn1 production. Xcn1 levels and expression of xcnA-L were increased in an ompR strain while Xcn2 levels and xcnMN expression were reduced. Xcn1 production was also increased in a strain lacking acetyl-phosphate that can donate phosphate groups to OmpR. Together these findings suggest that OmpR-phosphate negatively regulates xcnA-L gene expression while positively regulating xcnMN expression. HPLC-MS analysis revealed that Xcn1 was produced first and was subsequently converted to Xcn2. Inactivation of xcnM and xcnN eliminated conversion of Xcn1 to Xcn2 resulting in elevated Xcn1 production. The viability of the xcnM strain was reduced 20-fold relative to the wild-type strain supporting the idea that conversion of Xcn1 to Xcn2 provides a mechanism to avoid self-toxicity. Interestingly, inactivation of ompR enhanced cell viability during prolonged culturing.
Six novel linear peptides, named "rhabdopeptides", have been identified in the entomopathogenic bacterium Xenorhabdus nematophila after the discovery of the corresponding rdp gene cluster by using a promoter trap strategy for the detection of insect-inducible genes. The structures of these rhabdopeptides were deduced from labeling experiments combined with detailed MS analysis. Detailed analysis of an rdp mutant revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. Furthermore, two additional rhabdopeptide derivatives produced by Xenorhabdus cabanillasii were isolated, these showed activity against insect hemocytes thereby confirming the virulence of this novel class of compounds.
New methods are urgently needed to find novel natural products as structural leads for the development of new drugs against emerging diseases such as cancer and multiresistant bacterial infections. Here we introduce a reactivity-guided drug discovery approach for electrophilic natural products, a therapeutically relevant class of natural products that covalently modify their cellular targets, in crude extracts. Using carefully designed halogenated aromatic reagents, the process furnishes derivatives that are UV-active and highly conspicuous via mass spectrometry by virtue of an isotopically unique bromine or chlorine tag. In addition to the identification of high-value metabolites, the process facilitates the difficult task of structure elucidation by providing derivatives that are primed for X-ray crystallographic analysis. We show that a cysteine probe efficiently and chemoselectively labels enone-, β-lactam-, and β-lactone-based electrophilic natural products (parthenolide, andrographolide, wortmannin, penicillin G, salinosporamide), while a thiophenol probe preferentially labels epoxide-based electrophilic natural products (triptolide, epoxomicin, eponemycin, cyclomarin, salinamide). Using the optimized method, we were able to detect and isolate the epoxide-bearing natural product tirandalydigin from Salinispora and thereby link an orphan gene cluster to its gene product.
Feeding experiments have revealed that xenocoumacin I (1) is the precursor of xenocoumacin II (2), which was previously thought to be derived from the direct incorporation of proline. From mutational analyses of the biosynthesis gene cluster identified in the entomopathogenic bacterium Xenorhabdus nematophila, we propose that a desaturase (XcnN) and a saccharopine dehydrogenase‐like enzyme (XcnM) are essential for this unusual transformation.
Highlight describes the recently discovered prodrug activation mechanism found in the biosynthesis of nonribosomally produced peptides and peptide/polyketide hybrids as well as related mechanisms.
The biosynthesis gene cluster of the xenortides and a new derivative, xenortide D, which is produced in only trace amounts, was identified in Xenorhabdus nematophila. The structure of xenortide D was elucidated using a combination of labeling experiments followed by MS analysis and was confirmed by synthesis. Bioactivity tests revealed a weak activity of tryptamine-carrying xenortides against Plasmodium falciparum and Trypanosoma brucei.
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