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1.
Chem Commun (Camb) ; 56(22): 3329-3332, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083260

RESUMO

Taxodisones A and B, C30-terpenes with an unprecedented tetracyclo[12.4.0.0.2,709,14]octodecane core, were isolated from the seeds of Taxodium distichum. Their structures, including their configurations, were unambiguously determined. Their biomimetic synthesis suggests that they stem from diterpenes and monoterpenes, and not from squalene or oxidosqualene. In addition, their bioactivities were also evaluated.


Assuntos
Diterpenos/química , Taxodium/química , Biomimética , Catálise , Complexos de Coordenação/química , Cristalografia por Raios X , Reação de Cicloadição , Diterpenos/metabolismo , Érbio/química , Conformação Molecular , Sementes/química , Sementes/metabolismo , Taxodium/metabolismo , Terpenos/química , Terpenos/metabolismo
2.
Nat Commun ; 11(1): 971, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080175

RESUMO

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Diterpenos/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tripterygium/genética , Tripterygium/metabolismo , Abietanos/metabolismo , Antineoplásicos Fitogênicos/biossíntese , Vias Biossintéticas/genética , Medicamentos de Ervas Chinesas/metabolismo , Compostos de Epóxi/metabolismo , Perfilação da Expressão Gênica , Genoma de Planta , Humanos , Engenharia Metabólica , Metaboloma , Oxirredução , Filogenia , Plantas Medicinais/genética , Plantas Medicinais/metabolismo
3.
Talanta ; 209: 120591, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892095

RESUMO

Coffee is one of the most important commodities, showing sensitivity to environmental variations. The main effects and their interaction for two levels of atmospheric CO2 concentrations and two water regimes of a factorial design were investigated for the metabolic profiles of Coffea arabica raw beans using UV fingerprint analysis from a mixture design. UV fingerprint results obtained from pure ethanol and binary ethanol-dichloromethane mixtures showed the largest metabolic discriminations between CO2 levels and their extracts were investigated in detail. The biosynthesis of major metabolites, chlorogenic acids, cafestol, kahweol and caffeine were altered owing to environmental conditions. Higher amounts of chlorogenic acids and kahweol were observed in beans from unirrigated plants grown with enriched CO2 and irrigated ones at the current CO2 level. Water availability and CO2 concentration interaction affects the metabolite amounts. Besides a significant CO2 atmospheric effect water availability was a limiting factor for metabolite content only at current CO2 level, suggesting the successful metabolic coping of CO2 enriched Arabic coffee beans suffering future droughts.


Assuntos
Dióxido de Carbono/metabolismo , Coffea/metabolismo , Metaboloma , Sementes/metabolismo , Vias Biossintéticas , Cafeína/análise , Cafeína/metabolismo , Ácido Clorogênico/análise , Ácido Clorogênico/metabolismo , Coffea/química , Diterpenos/análise , Diterpenos/metabolismo , Secas , Sementes/química , Água/metabolismo
4.
Mol Biol Rep ; 47(2): 1507-1512, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31902054

RESUMO

Expression of genes involved in diterpene biosynthesis, especially momilactone and gibberellins (GAs), in rice plants (Oryza sativa L.) in response to barnyard grass (Echinochloa crus-galli) stress was examined. The three analyzed class II diterpene synthases had the highest fold change expression. Transcription patterns of genes for two homologs of momilactone synthases, OsMAS and OsMAS2, suggests their distinct roles in response to the presence of barnyard grass.


Assuntos
Diterpenos/metabolismo , Echinochloa/genética , Família Multigênica , Oryza/genética , Transcrição Genética , Vias Biossintéticas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Giberelinas/metabolismo
5.
Biosci Biotechnol Biochem ; 84(2): 347-357, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31670609

RESUMO

Bacillus ComQ participates in the biosynthesis of a quorum-sensing signaling molecule (ComX pheromone) through catalyzing the prenylation at a Trp residue of the precursor peptide (pre-ComX) with geranyl diphosphate (C10 type) or farnesyl diphosphate (C15 type). We hypothesized that several residues specifically conserved among either type of ComQs are important for their substrate specificities. Using a simple bioassay, we revealed that Phe63, Asn186, and Gly190 in ComQRO-E-2 (C10 type) were nondisplaceable to Ser63, Gly186, and Val190, the corresponding residues in the C15-type ComQ, respectively. A three-dimensional model suggested that the 186th and 190th residues are involved in the pre-ComX binding. In vitro analysis showed that substitution of Phe63 with Ser in ComQRO-E-2 significantly reduced the geranylation activity but substantially enhanced the farnesylation activity, whereas substitution of Ser63 with Phe in ComQ168 (C15 type) reduced the farnesylation activity. Therefore, the 63rd residue was found to be significant for the prenyl-substrate preference.Abbreviations: GPP: geranyl diphosphate; FPP: farnesyl diphosphate; IPP: isopentenyl diphosphate; GGPP: geranylgeranyl diphosphate; FARM: first aspartate-rich motif; SARM: second aspartate-rich motif; ß-Gal: ß-galactosidase; TBABG: tryptose blood agar base supplemented with glucose; X-gal: 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Bioensaio , Proteínas de Membrana/metabolismo , Percepção de Quorum , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Difosfatos/metabolismo , Diterpenos/metabolismo , Escherichia coli/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Prenilação de Proteína
6.
J Biotechnol ; 307: 29-34, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31689467

RESUMO

Yeast has been widely used for large-scale production of terpenoids. In yeast, modifications of terpenoid biosynthetic pathways have been intensively studied. tHMG1 (encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase of yeast) and UPC2-1 (the G888D mutant of UPC2 encoding a transcription factor) were integrated into yeast chromosome, and ERG9 (the squalene synthase gene of yeast) was knocked down to yield the chassis strain DH02. A F96C mutation in ERG20 (farnesyl diphosphate synthase of yeast) was conducted to obtain mERG20 which can function as a geranylgeranyl diphosphate synthase (GGPS). Then, three fused genes, including BTS1 (the yeast innate GGPS)-ERG20, ERG20-mERG20 and mERG20-ERG20, were constructed, and expressed either by the pESC-based plasmids in DH02, or by being integrated into DH02 chromosome. The highest geranylgeraniol (GGOH) content was observed in the extracts of DH12 integrated with ERG20-mERG20, corresponding to 3.2 and 2.3 folds of those of the strains integrated with BTS1 and mERG20, respectively. Finally, three genes encoding nor-copalyl diphosphate synthase (nor-CPS), ent-CPS and syn-CPS were integrated into the chromosome of DH12, respectively, to construct yeasts for producing corresponding copalyl diphosphates (CPPs). Thus, a yeast-based platform was built for characterizing all types of diterpene synthases using GGPP or various CPPs as their substrates.


Assuntos
Dimetilaliltranstransferase/metabolismo , Diterpenos/metabolismo , Geraniltranstransferase/metabolismo , Engenharia Metabólica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Substituição de Aminoácidos , Vias Biossintéticas , Dimetilaliltranstransferase/genética , Expressão Gênica , Fusão Gênica , Geraniltranstransferase/genética , Mutação , Fosfatos de Poli-Isoprenil/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-31779893

RESUMO

Class I TGA transcription factors (TFs) are known to participate in plant resistance responses, however, their regulatory functions in the biosynthesis of secondary metabolites were rarely revealed. In this study, a class I TGA TF, TwTGA1, from Tripterygium wilfordii Hook.f. was cloned and characterized. Overexpression of TwTGA1 in T. wilfordii Hook.f. cells increased the production of triptolide and two sesquiterpene pyridine alkaloids, which was further enhanced by methyl jasmonate (MeJA) treatment. RNA interference of TwTGA1 showed no significant effects on the production of these metabolites, indicating the existence of other TGA partner(s) with overlapping functions. Heterologous expression of TwTGA1 in tobacco By-2 cells promoted the biosynthesis of pyridine alkaloids. Under the elicitation of MeJA, the contents of nonpyrrolidine alkaloids further increased but not for nicotine. TwTGA1 could induce the expression of Putrescine N-methyltransferase (PMT) and N-methylputrescine oxidase 1 (MPO1) through binding to their promoters. Finally, transient expression of TwTGA1 in leaves of Catharanthus roseus changed both the profiles of vinca alkaloids (increased contents of serpentine and catharanthine, but decreased that of vinblastine) and the expressions of biosynthesis-related genes. The metabolic and transcriptional data indicated a relationship between jasmonic acid signaling pathway and the functions of TwTGA1.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Tripterygium/genética , Alcaloides/biossíntese , Sequência de Aminoácidos , Catharanthus/metabolismo , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Metabolismo Secundário , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tripterygium/metabolismo
8.
Mar Drugs ; 17(12)2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847481

RESUMO

Three new 11,20-epoxybriaranes-fragilides U-W (1-3), as well as two known metabolites, junceellonoid D (4) and junceellin (5), were obtained from the octocoral Junceella fragilis. The structures of briaranes 1-3 were elucidated by spectroscopic methods and briaranes 3 and 5 displayed inhibition effects on inducible nitric oxide synthase (iNOS) release from RAW264.7.


Assuntos
Antozoários/fisiologia , Diterpenos/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Diterpenos/química , Diterpenos/classificação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7
9.
Med Sci Monit ; 25: 8534-8543, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31714902

RESUMO

BACKGROUND Eriocalyxin B (EriB), a diterpenoid isolated from the plant Isodon eriocalyx, has been shown to possess anti-tumor properties. However, few systematic studies of the mechanism underlying the anti-tumor activity of Eriocalyxin B in prostate cancer cells have been published. The aim of this study was to investigate the effect of Eriocalyxin B on prostate cancer cells. MATERIAL AND METHODS In the present study, the PC-3 (androgen-independent) and 22RV1 (androgen-dependent) human prostate cancer cell lines were cultured with and without increasing doses of Eriocalyxin B. MTT assay was used to measure cell viability. Western blotting was performed to measure levels of proteins associated with apoptosis and autophagy. Flow cytometry was used to assess changes in cell apoptosis and cycle. Fluorescence microscopy was used to capture images of autophagy-related proteins. RESULTS Treatment of human prostate cancer cells with Eriocalyxin B resulted in apoptosis in a dose- and time-dependent manner. Eriocalyxin B also induced autophagy, with elevated LC3B-II protein expression and punctuate patterns. Additionally, autophagy protected prostate cancer cells from apoptosis induced by Eriocalyxin B, which was demonstrated by addition of chloroquine (CQ). Moreover, the results indicated that Eriocalyxin B could inhibit the phosphorylation of Akt and mTOR. Eriocalyxin B induced apoptosis and autophagy by inhibition of the Akt/mTOR pathway. CONCLUSIONS Eriocalyxin B induces apoptosis and autophagy involving the Akt/mTOR pathway in prostate cancer cells in vitro. These findings provide evidence for Eriocalyxin B as a potent therapeutic for the treatment of prostate cancer.


Assuntos
Diterpenos/metabolismo , Diterpenos/farmacologia , Neoplasias da Próstata/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , China , Humanos , Masculino , Células PC-3 , Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
10.
Molecules ; 24(21)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683764

RESUMO

Specialized plant terpenoids have found fortuitous uses in medicine due to their evolutionary and biochemical selection for biological activity in animals. However, these highly functionalized natural products are produced through complex biosynthetic pathways for which we have a complete understanding in only a few cases. Here we review some of the most effective and promising plant terpenoids that are currently used in medicine and medical research and provide updates on their biosynthesis, natural occurrence, and mechanism of action in the body. This includes pharmacologically useful plastidic terpenoids such as p-menthane monoterpenoids, cannabinoids, paclitaxel (taxol®), and ingenol mebutate which are derived from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, as well as cytosolic terpenoids such as thapsigargin and artemisinin produced through the mevalonate (MVA) pathway. We further provide a review of the MEP and MVA precursor pathways which supply the carbon skeletons for the downstream transformations yielding these medically significant natural products.


Assuntos
Vias Biossintéticas , Ácido Mevalônico/metabolismo , Monoterpenos/metabolismo , Terpenos/metabolismo , Animais , Canabinoides/metabolismo , Diterpenos/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Medicina Herbária , Humanos , Monoterpenos/uso terapêutico , Paclitaxel/metabolismo , Fosfatos Açúcares/metabolismo , Terpenos/uso terapêutico , Tapsigargina/metabolismo
11.
Nat Commun ; 10(1): 4850, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649252

RESUMO

Production of the anticancer drug Taxol and its precursors in heterologous hosts is more sustainable than extraction from tissues of yew trees or chemical synthesis. Although attempts to engineer the Taxol pathway in microbes have made significant progress, challenges such as functional expression of plant P450 enzymes remain to be addressed. Here, we introduce taxadiene synthase, taxadiene-5α-hydroxylase, and cytochrome P450 reductase in a high biomass plant Nicotiana benthamiana. Using a chloroplastic compartmentalized metabolic engineering strategy, combined with enhancement of isoprenoid precursors, we show that the engineered plants can produce taxadiene and taxadiene-5α-ol, the committed taxol intermediates, at 56.6 µg g-1 FW and 1.3 µg g-1 FW, respectively. In addition to the tools and strategies reported here, this study highlights the potential of Nicotiana spp. as an alternative platform for Taxol production.


Assuntos
Alcenos/metabolismo , Cloroplastos/metabolismo , Diterpenos/metabolismo , Engenharia Metabólica/métodos , Paclitaxel/biossíntese , Terpenos/metabolismo , Tabaco/metabolismo , Cloroplastos/genética , Sistema Enzimático do Citocromo P-450 , Vetores Genéticos , Isomerases/genética , Isomerases/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Taxoides/metabolismo , Taxus/genética , Tabaco/genética
12.
Commun Biol ; 2: 378, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31633069

RESUMO

Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Ritmo Circadiano/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Preparações Farmacêuticas/metabolismo , Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/genética , Aconitina/metabolismo , Aconitina/toxicidade , Animais , Linhagem Celular , Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Proteínas de Ligação a DNA/genética , Diterpenos/metabolismo , Diterpenos/toxicidade , Compostos de Epóxi/metabolismo , Compostos de Epóxi/toxicidade , Regulação Enzimológica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Inativação Metabólica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenantrenos/metabolismo , Fenantrenos/toxicidade , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Xenobióticos/metabolismo , Xenobióticos/toxicidade
13.
Org Biomol Chem ; 17(40): 8943-8957, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31482157

RESUMO

Diterpene pyrones (DTPs) are a group of well-known, mainly fungal, natural products, first isolated in 1966. As the name indicates, they are composed of two main structural features: a diterpenyl moiety and a pyrone ring. Various names have been given to this class of metabolites; however, biogenetic evidence indicates that they originate through the same metabolic pathway. Based on their biosynthesis, which leads to differences in their structural architecture, the DTPs can be classified into three main types. In addition to their intriguing chemistry, these compounds demonstrate a wide range of biological activities rendering them a desirable target for total synthesis. To date, sixty-seven DTPs have been isolated from various fungal species, with one example originating from the plant kingdom. This review aims at unifying the classification of these compounds, in addition to presenting a detailed description of their isolation, bioactivities, biosynthesis, and total synthesis.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Pironas/química , Pironas/metabolismo , Produtos Biológicos/síntese química , Diterpenos/síntese química , Estrutura Molecular , Pironas/síntese química
14.
Mol Biol Rep ; 46(6): 6311-6323, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31535324

RESUMO

Norisoprenoids are produced from carotenoids under oxidative degradation mediated by carotenoid cleavage dioxygenases (CCDs) and contribute to floral and fruity notes in grape berries and wine. The diversity of CCD substrates and products has been demonstrated by in vitro recombinant proteins extracted from Escherichia coli expressing CCD genes and of in vivo proteins in an E. coli system co-expressing genes for carotenoid synthesis and cleavage. In the current study, VvCCD1 and VvCCD4b were isolated from the cDNA library of Vitis vinifera L. cv. Cabernet Sauvignon and then transformed into carotenoid-accumulating recombinant Saccharomyces cerevisiae strains. The expression of the target genes was monitored during the yeast growth period, and the accumulation of carotenoids and norisoprenoids in the recombinant strains was measured. The results indicated that both of the VvCCDs cleaved ß-carotene at the 7, 8 (7', 8') position into ß-cyclocitral for the first time. Additionally, the two enzymes also degraded ß-carotene at the 9, 10 (9', 10') position to generate ß-ionone and cleaved lycopene at the 5, 6 (5', 6') position into 6-methyl-5-hepten-2-one. These findings suggested that the VvCCDs may possess more cleavage characteristics under the eukaryotic expression system in S. cerevisiae than the prokaryotic system in E. coli, which could better explain the biochemical functions of VvCCDs in grape berries.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Saccharomyces cerevisiae/genética , Vitis/enzimologia , Aldeídos/metabolismo , Clonagem Molecular , Diterpenos/metabolismo , Biblioteca Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Transformação Genética , Vitis/genética , beta Caroteno/metabolismo
15.
Chin J Nat Med ; 17(8): 575-584, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472894

RESUMO

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.


Assuntos
Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Domínio Catalítico , Compostos de Epóxi/metabolismo , Hidroximetilglutaril-CoA Sintase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Interferência de RNA , Terpenos/metabolismo , Tripterygium/enzimologia , Tripterygium/genética , Triterpenos/metabolismo
16.
Phytochemistry ; 167: 112082, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421542

RESUMO

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ±â€¯0.05 µg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ±â€¯0.001 µg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.


Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Diterpenos/metabolismo , Engenharia Genética , Alquil e Aril Transferases/genética , Cloroplastos/genética , Cistus/enzimologia , Cistus/genética , Diterpenos/química , Proteínas de Plantas/genética , Transformação Genética
17.
Int J Mol Sci ; 20(15)2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31382458

RESUMO

Channelrhodopsin-2 (ChR2) is a light-activated and non-selective cationic channel protein that can be easily expressed in specific neurons to control neuronal activity by light. Although ChR2 has been extensively used as an optogenetic tool in neuroscience research, the molecular mechanism of cation channel formation following retinal photoisomerization in ChR2 is not well understood. In this paper, studies of the closed and opened state ChR2 structures are presented. The formation of the cationic channel is elucidated in atomic detail using molecular dynamics simulations on the all-trans-retinal (ChR2-trans) configuration of ChR2 and its isomerization products, 13-cis-retinal (ChR2-cis) configuration, respectively. Photoisomerization of the retinal-chromophore causes the destruction of interactions among the crucial residues (e.g., E90, E82, N258, and R268) around the channel and the extended H-bond network mediated by numerous water molecules, which opens the pore. Steering molecular dynamics (SMD) simulations show that the electrostatic interactions at the binding sites in intracellular gate (ICG) and central gate (CG) can influence the transmembrane transport of Na+ in ChR2-cis obviously. Potential of mean force (PMF) constructed by SMD and umbrella sampling also found the existing energy wells at these two binding sites during the transportation of Na+. These wells partly hinder the penetration of Na+ into cytoplasm through the ion channel. This investigation provides a theoretical insight on the formation mechanism of ion channels and the mechanism of ion permeation.


Assuntos
Channelrhodopsins/metabolismo , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Channelrhodopsins/química , Chlamydomonas reinhardtii/química , Diterpenos/química , Diterpenos/metabolismo , Transporte de Íons , Isomerismo , Simulação de Dinâmica Molecular , Proteínas de Plantas/química , Conformação Proteica , Multimerização Proteica , Retinaldeído/química , Retinaldeído/metabolismo
18.
Plant Physiol Biochem ; 142: 405-414, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31408844

RESUMO

Chamaecyparis formosensis (Cupressaceae) is among the most precious endemic conifers in Taiwan. Field study was conducted on seasonal variations in emission rates and compositions of terpenoids from this tree species of two different ages. A total of 21 terpenoids were detected, of which there were 13 monoterpenoids (MTs), 4 sesquiterpenoids (STs), and 4 diterpenoids (DTs). MTs dominated the emissions in both saplings and adult trees and produced more than 80% of terpene emissions. Contrasting seasonal pattern between saplings and adult trees was found. Total actual emissions from saplings were higher in cold seasons (range, 64.40 ±â€¯13.18 to 140.74 ±â€¯18.90 ng g-1 h-1) than in warm seasons (range, 55.63 ±â€¯15.84 to 63.48 ±â€¯11.85 ng g-1 h-1). Photosynthetically active radiation (PAR) was found to be the most important factor affecting terpene emissions from saplings. On the contrary, higher emissions were found in warm seasons for adult trees (range, 101.49 ±â€¯12.29 to 181.35 ±â€¯80.15 ng g-1 h-1), and the emissions were mainly in response to temperature. Some compounds in C. formosensis of both ages (e.g., ß-myrcene, α-terpinene, trans-ß-ocimene, terpinen-4-ol, α-cedrene and trans-ß-farnesene) showed comparably higher contents in cold seasons. Results presented here provide important fundamental information for better understanding of forest bathing and estimating air quality in Taiwan.


Assuntos
Chamaecyparis/metabolismo , Terpenos/metabolismo , Envelhecimento , Chamaecyparis/fisiologia , Diterpenos/metabolismo , Monoterpenos/metabolismo , Fotossíntese/fisiologia , Estações do Ano , Taiwan , Temperatura
19.
Planta ; 250(5): 1613-1620, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31388830

RESUMO

MAIN CONCLUSION: A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA9to GA20, but not GA4to GA1. Gibberellins (GAs) play essential roles in plant growth and development. Previous characterization of GA20- and GA3-oxidases yielded a large number of genetic elements that can interconvert different GAs. However, enzymes that catalyze the 13-hydroxylation step are rarely identified. Here, we report that the GA13-oxidase of Tripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase instead of reported cytochrome P450 oxygenases, among 376 differential proteins in comparative proteomics. Phylogenetic analysis showed that the enzyme resides in its own independent branch in the DOXC class. Unexpectedly, it specifically catalyzes the conversion of GA9 to GA20, but not GA4 to GA1. Contrary to the previous research, TwGA13ox transcriptional expression was upregulated ~ 146 times by exogenous application of methyl jasmonate (MeJA). RNAi targeting of TwGA13ox in T. wilfordii led to an 89.9% decrease of triptolide, a diterpenoid epoxide with extensive anti-inflammatory and anti-tumor properties. In subsequent MeJA supplementation experiments, triptolide production increased 13.4-times. TwGA13ox displayed root-specific expression. Our results provide a new GA13-oxidase from plants and elucidate the metabolic associations within the diterpenoid biosynthetic pathway (GAs, triptolide) at the genetic level.


Assuntos
Acetatos/farmacologia , Ciclopentanos/farmacologia , Dioxigenases/metabolismo , Regulação Enzimológica da Expressão Gênica , Giberelinas/metabolismo , Oxirredutases/metabolismo , Oxilipinas/farmacologia , Tripterygium/enzimologia , Vias Biossintéticas , Dioxigenases/genética , Diterpenos/metabolismo , Compostos de Epóxi/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oxirredutases/genética , Fenantrenos/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tripterygium/genética
20.
Phytochemistry ; 166: 112062, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31299395

RESUMO

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.


Assuntos
Biocatálise , Diterpenos/química , Diterpenos/metabolismo , Glucosídeos/química , Glucosiltransferases/metabolismo , Tripterygium/química , Glucosiltransferases/química , Simulação de Acoplamento Molecular , Conformação Proteica
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