RESUMO
Enzymes involved in secondary metabolite biosynthetic pathways have typically evolutionarily diverged from their counterparts functioning in primary metabolism. They often catalyze diverse and complex chemical transformations and are thus a treasure trove for the discovery of unique enzyme-mediated chemistries. Besides major natural product classes, such as terpenoids, polyketides, and ribosomally or nonribosomally synthesized peptides, biosynthetic investigations of noncanonical natural product biosynthetic pathways often reveal functionally distinct enzyme chemistries. In this Perspective, we aim to highlight challenges and opportunities of biosynthetic investigations on noncanonical natural product pathways that utilize primary metabolites as building blocks, otherwise generally considered as enzyme cofactors. A focus is made on the discovered chemical and enzymological novelties.
RESUMO
ABSTRATCT: ß-Nicotinamide adenine dinucleotide (ß-NAD) is a pivotal metabolite for all living organisms and functions as a diffusible electron acceptor and carrier in the catabolic arms of metabolism1,2. Furthermore, ß-NAD is involved in diverse epigenetic, immunological and stress-associated processes, where it is known to be sacrificially utilized as an ADP-ribosyl donor for protein and DNA modifications, or the generation of cell-signalling molecules3,4. Here we report the function of ß-NAD in secondary metabolite biosynthetic pathways, in which the nicotinamide dinucleotide framework is heavily decorated and serves as a building block for the assembly of a novel class of natural products. The gatekeeping enzyme of the discovered pathway (SbzP) catalyses a pyridoxal phosphate-dependent [3+2]-annulation reaction between ß-NAD and S-adenosylmethionine, generating a 6-azatetrahydroindane scaffold. Members of this novel family of ß-NAD-tailoring enzymes are widely distributed in the bacterial kingdom and are encoded in diverse biosynthetic gene clusters. The findings of this work set the stage for the discovery and exploitation of ß-NAD-derived natural products.
Assuntos
Produtos Biológicos , NAD , Catálise , NAD/metabolismo , Niacinamida , Transdução de SinaisRESUMO
Sulfonamides and sulfamates are a group of organosulfur compounds that contain the signature sulfamoyl structural motif. These compounds were initially only known as synthetic antibacterial drugs but were later also discovered as natural products. Eight highly potent examples have been isolated from actinomycetes to date, illustrating the large biosynthetic repertoire of this bacterial genus. For the biosynthesis of these compounds, several distinct and unique biosynthetic machineries have been discovered, capable to generate the unique S-N bond. For the creation of novel, second generation natural products by biosynthetic engineering efforts, a detailed understanding of the underlying enzyme machinery toward potent structural motifs is crucial. In this review, we aim to summarize the current state of knowledge on sulfonamide and sulfamate biosynthesis. A detailed discussion for the secondary sulfamate ascamycin, the tertiary sulfonamide sulfadixiamycin A, and the secondary sulfonamide SB-203208 is provided and their bioactivities and mode of actions are discussed.
Assuntos
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Sulfonamidas/metabolismo , Ácidos Sulfônicos/metabolismo , Actinobacteria/química , Actinobacteria/genética , Antibacterianos/química , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Sulfonamidas/química , Ácidos Sulfônicos/químicaRESUMO
Covering: up to July 2020Fungal meroterpenoid cyclases are a recently discovered emerging family of membrane-integrated, non-canonical terpene cyclases. They catalyze the conversion of hybrid isoprenic precursors towards complex scaffolds and are therefore of great importance in the structure diversification in meroterpenoid biosynthesis. The products of these pathways exhibit intriguing molecular scaffolds and highly potent bioactivities, making them privileged structures from Nature and attractive candidates for drug development or industrial applications. This review will provide a comprehensive and comparative view on fungal meroterpenoid cyclases, their intriguing chemistries and importance for the scaffold formation step towards polycyclic meroterpenoid natural products.
Assuntos
Carboxiliases/química , Carboxiliases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Alcenos/química , Alcenos/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Fenóis/química , Fenóis/metabolismo , Filogenia , Piridinas/química , Piridinas/metabolismo , Pironas/química , Pironas/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Terpenos/química , Terpenos/metabolismoRESUMO
Fungal meroterpenoids are a diverse group of hybrid natural products with impressive structural complexity and high potential as drug candidates. In this work, we evaluate the promiscuity of the early structure diversity-generating step in fungal meroterpenoid biosynthetic pathways: the multibond-forming polyene cyclizations catalyzed by the yet poorly understood family of fungal meroterpenoid cyclases. In total, 12 unnatural meroterpenoids were accessed chemoenzymatically using synthetic substrates. Their complex structures were determined by 2D NMR studies as well as crystalline-sponge-based X-ray diffraction analyses. The results obtained revealed a high degree of enzyme promiscuity and experimental results which together with quantum chemical calculations provided a deeper insight into the catalytic activity of this new family of non-canonical, terpene cyclases. The knowledge obtained paves the way to design and engineer artificial pathways towards second generation meroterpenoids with valuable bioactivities based on combinatorial biosynthetic strategies.
Assuntos
Vias Biossintéticas/genética , Fungos/química , Terpenos/químicaRESUMO
A biomimetic route to farnesyl pyrophosphate and dimethyl orsellinic acid (DMOA)-derived meroterpenoid scaffolds has yet to be reported despite great interest from the chemistry and biomedical research communities. A concise synthetic route with the potential to access DMOA-derived meroterpenoids is highly desirable to create a library of related compounds. Herein, we report novel dearomatization methodology followed by polyene cyclization to access DMOA-derived meroterpenoid frameworks in six steps from commercially available starting materials. Furthermore, several farnesyl alkene substrates were used to generate structurally novel, DMOA-derived meroterpenoid derivatives. DFT calculations combined with experimentation provided a rationale for the observed thermodynamic distribution of polycyclization products.
Assuntos
Biomimética/métodos , Polienos/química , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Terpenos/metabolismo , CiclizaçãoRESUMO
The skeletons of some classes of terpenoids are unusual in that they contain a larger number of Me groups (or their biosynthetic equivalents such as olefinic methylene groups, hydroxymethyl groups, aldehydes, or carboxylic acids and their derivatives) than provided by their oligoprenyl diphosphate precursor. This is sometimes the result of an oxidative ring-opening reaction at a terpene-cyclase-derived molecule containing the regular number of Me group equivalents, as observed for picrotoxan sesquiterpenes. In this study a sesquiterpene cyclase from Trichoderma spp. is described that can convert farnesyl diphosphate (FPP) directly via a remarkable skeletal rearrangement into trichobrasilenol, a new brasilane sesquiterpene with one additional Me group equivalent compared to FPP. A mechanistic hypothesis for the formation of the brasilane skeleton is supported by extensive isotopic labelling studies.
Assuntos
Carbono-Carbono Liases/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Trichoderma/metabolismo , Carbono-Carbono Liases/química , Carbono-Carbono Liases/genética , Estrutura Molecular , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/química , Estereoisomerismo , Trichoderma/enzimologia , Trichoderma/genéticaRESUMO
Plant pathogenic fungi in the Fusarium genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across Fusarium have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs) across publicly available genomes from Fusarium, to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs) to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to Fusarium pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fusarium oxysporum Fo47, while being absent in that of the tomato pathogen F. oxysporum f.sp. lycopersici. Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the Fusarium poae supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in Fusarium paves the way for targeted natural product discovery based on automated identification of species-specific pathways as well as for connecting species ecology to the taxonomic distributions of BGCs.
RESUMO
Analysis of the volatile terpenes produced by seven fungal strains of the genus Trichoderma by use of a closed-loop stripping apparatus (CLSA) revealed a common production of harzianone, a bioactive, structurally unique diterpenoid consisting of a fused tetracyclic 4,7,5,6-membered ring system. The terpene cyclization mechanism was studied by feeding experiments using selectively 13 C- and 2 H-labeled synthetic mevalonolactone isotopologues, followed by analysis of the incorporation patterns by 13 Câ NMR spectroscopy and GC/MS. The structure of harzianone was further supported from a 13 C,13 Câ COSY experiment of the in-vivo-generated fully 13 C-labeled diterpene.
Assuntos
Diterpenos/metabolismo , Trichoderma/metabolismo , Isótopos de Carbono/química , Ciclização , Diterpenos/química , Cromatografia Gasosa-Espectrometria de Massas , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/síntese química , Ácido Mevalônico/química , Conformação Molecular , Estereoisomerismo , Terpenos/química , Terpenos/metabolismo , Trichoderma/química , Trítio/químicaRESUMO
The volatiles emitted by five fungal strains previously isolated from the marine sponge Callyspongia cf. flammea were captured with a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Besides several widespread compounds, a series of metabolites with interesting bioactivities were found, including the quorum sensing inhibitor protoanemonin, the fungal phytotoxin 3,4-dimethylpentan-4-olide, and the insect attractant 1,2,4-trimethoxybenzene. In addition, the aromatic polyketides isotorquatone and chartabomone that are both known from Eucalyptus and a new O-desmethyl derivative were identified. The biosynthesis of isotorquatone was studied by feeding experiments with isotopically labeled precursors and its absolute configuration was determined by enantioselective synthesis of a reference compound. Bioactivity testings showed algicidal activity for some of the identified compounds, suggesting a potential ecological function in sponge defence.
Assuntos
Callyspongia/microbiologia , Micobioma , Compostos Orgânicos Voláteis/química , Animais , Callyspongia/metabolismo , Estrutura Molecular , Compostos Orgânicos Voláteis/metabolismoRESUMO
The biosynthetic gene cluster for the antiplasmodial natural product siphonazole was identified by using a combination of genome mining, imaging, and expression studies in the natural producer Herpetosiphon sp. B060. The siphonazole backbone is assembled from an unusual starter unit from the shikimate pathway that is extended by the action of polyketide synthases and non-ribosomal peptide synthetases with unusual domain structures, including several split modules and a large number of duplicated domains and domains predicted to be inactive. Product release proceeds through decarboxylation and dehydration independent of the thioesterase SphJ and yields the diene terminus of siphonazole. High variation in terms of codon-usage within the gene cluster, together with the dislocated domain organization, suggest a recent emergence in evolutionary terms.
Assuntos
Antimaláricos/metabolismo , Produtos Biológicos/metabolismo , Chloroflexi/genética , Oxazóis/metabolismo , Antimaláricos/química , Antimaláricos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Chloroflexi/metabolismo , Mineração de Dados , Descarboxilação , Espectrometria de Massas , Família Multigênica , Oxazóis/química , Oxazóis/isolamento & purificaçãoRESUMO
Three sesquiterpene cyclases from Streptomyces scabei 87.22, Streptomyces venezuelae ATCC 10712 and Streptomyces clavuligerus ATCC 27064 were characterised and their products were identified as (-)-neomeranol B, (+)-isodauc-8-en-11-ol and (+)-intermedeol, respectively. The stereochemical courses of the terpene cyclisations were investigated by use of various (13)C-labelled FPP isotopomers. A quick and easy test was developed that allows to distinguish reprotonations of olefinic double bonds in neutral intermediates from the two stereoheterotopic faces. The method makes use of incubating (13)C-FPP isotopomers labelled at the reprotonated carbon in deuterium oxide and subsequent HSQC analysis of the product. A 1,7-cyclisation towards (+)-isodauc-8-en-11-ol was followed by use of (1,7-(13)C2)FPP. Surprisingly, the (+)-isodauc-8-en-11-ol also accepted (2Z,6E)-FPP resulting in the same product profile as obtained from (2E,6E)-FPP.
Assuntos
Terpenos/química , Ciclização , Conformação Molecular , EstereoisomerismoRESUMO
An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen ((13)C1)FPP isotopomers (FPP=farnesyl diphosphate) and ((13)C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including (13)C, (13)Câ COSY experiments. The ((13)C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.
Assuntos
Alquil e Aril Transferases/metabolismo , Sesquiterpenos de Germacrano/química , Temperatura , Alquil e Aril Transferases/química , Isótopos de Carbono , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Conformação Molecular , Fosfatos de Poli-Isoprenil/síntese química , Fosfatos de Poli-Isoprenil/química , Sesquiterpenos/síntese química , Sesquiterpenos/química , Sesquiterpenos de Germacrano/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Feeding of (2,3,4,5,6-(13) C5 )mevalonolactone to the fungus Hypomyces odoratus resulted in a completely labeled sesquiterpene ether. The connectivity of the carbon atoms was easily deduced from a (13) C,(13) Câ COSY spectrum, revealing a structure that was different from the previously reported structure of hypodoratoxide, even though the reported (13) Câ NMR data matched. A structural revision of hypodoratoxide is thus presented. Its absolute configuration was tentatively assigned from its co-metabolite cis-dihydroagarofuran. Its biosynthesis was investigated by feeding of (3-(13) C)- and (4,6-(13) C2 )mevalonolactone, which gave insights into the complex rearrangement of the carbon skeleton during terpene cyclization by analysis of the (13) C,(13) C couplings.
Assuntos
Sesquiterpenos/metabolismo , Ascomicetos/metabolismo , Isótopos de Carbono/química , Espectroscopia de Ressonância Magnética , Ácido Mevalônico/análogos & derivados , Ácido Mevalônico/química , Ácido Mevalônico/metabolismo , Conformação Molecular , Sesquiterpenos/químicaRESUMO
The volatiles released by agar plate cultures of nineteen actinomycetes whose genomes were recently sequenced were collected by use of a closed-loop stripping apparatus (CLSA) and analysed by GC/MS. In total, 178 compounds from various classes were identified. The most interesting findings were the detection of the insect pheromone frontalin in Streptomyces varsoviensis, and the emission of the unusual plant metabolite 1-nitro-2-phenylethane. Its biosynthesis from phenylalanine was investigated in isotopic labelling experiments. Furthermore, the identified terpenes were correlated to the information about terpene cyclase homologs encoded in the investigated strains. The analytical data were in line with functionally characterised bacterial terpene cyclases and particularly corroborated the recently suggested function of a terpene cyclase from Streptomyces violaceusniger by the identification of a functional homolog in Streptomyces rapamycinicus.
Assuntos
Actinobacteria/genética , Compostos Orgânicos Voláteis/isolamento & purificação , Actinobacteria/química , Actinobacteria/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Streptomyces/química , Streptomyces/metabolismo , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
Six (13)C-labelled isotopomers of mevalonolactone were synthesised and used in feeding experiments with the endophytic fungus Geniculosporium. The high incorporation rates of (13)C-label into a sesquiterpene that was found in headspace extracts of the fungus enabled unambiguous identification of this volatile as pogostol without the need for compound purification, simply by collecting the volatile fraction with a closed-loop stripping apparatus followed by direct (13)C NMR analysis (CLSA-NMR). The feeding experiments also gave insights into the biosynthesis of pogostol, including stereochemical aspects of the terpene cyclisation reaction. The possible biological function of pogostol is discussed.