RESUMEN
Innovative discovery approaches such as genome-mining and metabolomics-inspired methods have reshaped the natural product research field, complementing traditional bioactivity-based screens and allowing hitherto unseen compounds to be uncovered from previously investigated producers. In line with these trends, we report here imidacins, a novel class of secondary metabolites specific to the myxobacterial genus Stigmatella. A combination of secondary metabolome analysis, genome-mining techniques, spectroscopic analysis, and finally total synthesis was used to allow structure elucidation. Imidacins are urocanate-derived aliphatic acids with an adjacent cyclopropane moiety, structural features unprecedented in natural products to date.
Asunto(s)
Stigmatella aurantiaca , Estructura Molecular , Stigmatella aurantiaca/química , Alcaloides/química , Alcaloides/síntesis química , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/síntesis química , Myxococcales/química , Ciclopropanos/química , Ciclopropanos/farmacología , Ciclopropanos/síntesis químicaRESUMEN
The myxocoumarins A and B from Stigmatella aurantiaca MYX-030 are natural products featuring unusual nitro- and long-chain alkyl substitution. While myxocoumarin A was shown to exhibit strong antifungal properties, the antifungal potential of myxocoumarin B was not yet assessed due to low production titers during initial isolation. We therefore developed a total synthesis of myxocoumarin B that involves a late-stage Pd-catalyzed nitration of the coumarin core. The availability of synthetic material facilitated the initial evaluation of the bioactivity of myxocoumarin B, which revealed a lack of activity against medically relevant Candida sp. and low cytotoxicity in vitro against human fibroblasts (MRC-5) and in vivo (zebrafish).
Asunto(s)
Antifúngicos/farmacología , Ascomicetos/efectos de los fármacos , Productos Biológicos/farmacología , Botrytis/efectos de los fármacos , Cumarinas/farmacología , Fibroblastos/efectos de los fármacos , Fusarium/efectos de los fármacos , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Productos Biológicos/síntesis química , Productos Biológicos/química , Cumarinas/síntesis química , Cumarinas/química , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Stigmatella aurantiaca/química , Relación Estructura-Actividad , Pez Cebra/embriologíaRESUMEN
Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.
Asunto(s)
Actinobacteria/enzimología , Productos Biológicos/metabolismo , Stigmatella aurantiaca/enzimología , Fosfatos de Azúcar/metabolismo , Actinobacteria/química , Actinobacteria/genética , Actinobacteria/metabolismo , Productos Biológicos/química , Biología Computacional , Genes Bacterianos , Inositol/análogos & derivados , Inositol/química , Inositol/metabolismo , Modelos Moleculares , Filogenia , Stigmatella aurantiaca/química , Stigmatella aurantiaca/genética , Stigmatella aurantiaca/metabolismo , Fosfatos de Azúcar/químicaRESUMEN
The isolation of aurachins A-L (1-11) from Stigmatella aurantiaca strain Sg a15 is described. Their structures and relative configurations were deduced from spectroscopic data, in particular NMR. Three structural types were identified: A-type aurachins (1, 2, 6) are C-3 oxygen-substituted quinolines carrying a farnesyl residue on C-4, C-type aurachins (3, 4, 7-11) are C-4 oxygen-substituted quinolines carrying a farnesyl residue on C-3, and C-type aurachin E (5) has a [1,1a,8,d]imidazoloquinoline structure. Feeding of (13)C-labeled precursors showed that the quinoline ring is constructed from anthranilic acid and acetate, and the farnesyl residue from acetate by both the mevalonate and nonmevalonate pathways. Further, feeding of labeled aurachin C (3) indicated the A-type aurachins are derived by a novel intramolecular 3,4-migration of the farnesyl residue that is induced by a 2,3-epoxidation and terminated by a reduction step. (18)O-Labeling experiments indicated the new oxygen substituents originate from atomospheric oxygen. On the basis of these results a biosynthetic scheme covering all aurachins is proposed. It is further proposed that quinolones with an unorthodox substitution pattern, such as the 2-geranylquinolones from Pseudonocardia sp. and the 3-heptylquinolones from Pseudomonas sp., are formed by related rearrangement mechanisms.
Asunto(s)
Stigmatella aurantiaca/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Oxígeno/química , Quinolinas/química , Quinolinas/aislamiento & purificación , Quinolinas/metabolismo , Quinolonas/química , Quinolonas/aislamiento & purificación , Stigmatella aurantiaca/metabolismoRESUMEN
Myxochromides are cyclic depsipeptides with an unsaturated polyketide side chain, which have been reported from different myxobacterial species, e.g., Myxococcus xanthus and Stigmatella aurantiaca. To date, myxochromides are subdivided into the groups A and S, according to their peptidic core structure. The peptide moiety of the new myxochromide B3 (1), which was isolated from a myxobacterial strain of the genus Myxococcus, differs from that of myxochromides A and S. Compound 1 thus is the first representative of a new group of myxochromides. For myxochromide A3 (2) the complete and assigned spectroscopic data are described. For the structure elucidation one- and two-dimensional NMR spectroscopy as well as mass spectrometry have been applied. Configurational analysis has been accomplished by chiral GC-MS and HPLC.
Asunto(s)
Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Myxococcus xanthus/química , Microbiología del Suelo , Stigmatella aurantiaca/química , Francia , Estructura Molecular , Myxococcus xanthus/genética , Resonancia Magnética Nuclear Biomolecular , Stigmatella aurantiaca/genéticaRESUMEN
The myxochelins are catecholate-type siderophores produced by a number of myxobacterial strains, and their corresponding biosynthetic gene clusters have been identified in Stigmatella aurantiaca Sg a15, and Sorangium cellulosum So ce56; the latter being presented in this work. Biochemical and genetic studies described here further clarify myxochelin biosynthesis. In addition to the myxochelin A biosynthetic complex, the aminotransferase MxcL is required in order to form myxochelin B, starting from 2,3-dihydroxy benzoic acid and L-lysine. Additionally, the substrate specificity of the myxochelin A biosynthetic complex was analyzed in vitro; this led to the formation of novel myxochelin derivatives. Furthermore, MxcD was over-expressed and its function as an active isochorismic acid synthase in Escherichia coli was verified by complementation studies, as was activity in vitro. The organization of the myxochelin gene cluster of S. cellulosum So ce56 was compared to that of the Sg a15 gene cluster. The comparison revealed that although the organization of the biosynthetic genes is completely different, the biosynthesis is most probably extremely similar.
Asunto(s)
Lisina/análogos & derivados , Myxococcales/química , Sideróforos/biosíntesis , Catecoles/química , Genes Bacterianos , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Lisina/biosíntesis , Lisina/química , Estructura Molecular , Familia de Multigenes , Myxococcales/genética , Myxococcales/metabolismo , Sideróforos/química , Stigmatella aurantiaca/química , Stigmatella aurantiaca/genética , Stigmatella aurantiaca/metabolismoRESUMEN
The myxobacterium Stigmatella aurantiaca DW4/3-1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1-3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Stigmatella aurantiaca/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Molecular , Familia de Multigenes , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Stigmatella aurantiaca/química , Stigmatella aurantiaca/genética , Especificidad por SustratoRESUMEN
A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain alpha-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.
Asunto(s)
Acilcoenzima A/biosíntesis , Ácido Mevalónico/metabolismo , Myxococcales/metabolismo , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Animales , Glutaratos/química , Glutaratos/metabolismo , Leucina/metabolismo , Metacrilatos , Ácido Mevalónico/química , Estructura Molecular , Myxococcales/química , Quinolinas/química , Quinolinas/metabolismo , Stigmatella aurantiaca/química , Stigmatella aurantiaca/genética , Stigmatella aurantiaca/metabolismo , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Today, many valuable proteins can be obtained in sufficient amounts using recombinant DNA techniques. However, frequently the expression of recombinant proteins results in the accumulation of the product in dense amorphous deposits inside the cells, called inclusion bodies. The challenge then is to transform these inactive and misfolded protein aggregates into soluble bioactive forms. Although a number of general guidelines have been proposed, the search for proper reconstitution conditions can be very laborious and time consuming. Here, we suggest a new versatile approach for solubilization and refolding of inclusion body proteins using a water-sodium bis-2-ethylhexyl sulfosuccinate-isooctane reverse micellar system. Instead of amorphous aggregates, a transparent solution is obtained, where refolded protein is entrapped inside the micelles. The entrapped enzyme has native-like secondary structure and catalytic activity. This approach has been implemented with Fusarium galactose oxidase and Stigmatella aurantiaca putative galactose oxidase.