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1.
Food Chem ; 339: 128151, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33152896

RESUMO

The effects of aloe vera (Aloe vera (L.) Burm. f.) gel treatment on the incidence of superficial scald in 'Starking' apples (Malus domestica Borkh. Var. Starking) during cold storage were studied. Apples were harvested at the pre-climacteric stage and treated with aloe vera gel. The treatment increased malondialdehyde content and membrane lipid damage. Furthermore, it inhibited the release of ethylene at the early stage but increased it in the later stage. The expression level of ACC synthase 1 (MdACS1) also increased, and the antioxidant capacity in apples, particularly, catalase, peroxidase, and superoxide dismutase activities, all decreased, while concomitantly, the content of α-farnesene and its oxidation product, conjugated triene increased, thereby aggravating superficial scald incidence during storage at low temperature.


Assuntos
Conservação de Alimentos/métodos , Malus/fisiologia , Preparações de Plantas , Antioxidantes/metabolismo , Temperatura Baixa , Ciclopropanos/farmacologia , Enzimas/metabolismo , Etilenos/metabolismo , Armazenamento de Alimentos , Frutas/fisiologia , Malus/efeitos dos fármacos , Oxirredução , Preparações de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo
2.
PLoS One ; 15(12): e0235028, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362256

RESUMO

Cyclocephalini beetles of the genus Cyclocephala (Coleoptera: Melolonthidae: Dynastinae) use flowers of some plants as food, shelter, and mating sites. However, little is known about floral scent chemistry involved in this interaction. Here we show that a sesquiterpene alcohol mediates attraction of Cyclocephala paraguayensis Arrow, on bottle gourd flowers, Lagenaria siceraria (Cucurbitaceae). Both males and females started to aggregate on the flowers at twilight; after that, mating began and remained for the entire night. GC-FID/EAD analysis of the L. siceraria floral scent collected in the field revealed that only the major constituent of the airborne volatiles elicited electroantennographic responses on male and female antennae of C. paraguayensis. This compound was identified as (3S,6E)-nerolidol, which was tested in two field trapping trials in Brazil. In the first bioassay, traps baited with nerolidol (mix of isomers) captured significantly more adult C. paraguayensis than control traps. In the second field trial, catches in traps baited with a mixture of isomers or enantiopure nerolidol were significantly higher than captures in control traps, but the treatments did not differ significantly. Analysis from the gut content of adult C. paraguayensis showed the presence of pollen, suggesting that they also use bottle gourd flowers for their nourishment. Taken together, these results suggest that (3S,6E)-nerolidol plays an essential role in the reproductive behavior of C. paraguayensis by eliciting aggregation, mating, and feeding.


Assuntos
Besouros/fisiologia , Cucurbita/metabolismo , Sesquiterpenos/metabolismo , Animais , Brasil , Besouros/metabolismo , Cucurbita/fisiologia , Cucurbitaceae/metabolismo , Cucurbitaceae/fisiologia , Feminino , Flores/fisiologia , Masculino , Odorantes/análise , Feromônios/análise , Pólen/fisiologia , Polinização , Sesquiterpenos/análise
3.
PLoS One ; 15(10): e0239893, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33035251

RESUMO

The ecdysteroid and sesquiterpenoid pathways control growth, developmental transition, and embryogenesis in insects. However, the function of orthologous genes and the cross-talk between both pathways remain largely uncharacterized in non-insect arthropods. Spook (Spo) and Juvenile hormone acid o-methyltransferase (Jhamt) have been suggested to function as rate-limiting factors in ecdysteroid and sesquiterpenoid biosynthesis, respectively, in insects. In this study, we report on the functions of Spo and Jhamt and the cross-talk between them in embryos of the branchiopod crustacean Daphnia magna. Spo expression was activated at the onset of gastrulation, with the depletion of Spo transcript by RNAi resulting in developmental arrest at this stage. This phenotype could be partially rescued by supplementation with 20-hydroxyecdysone, indicating that Spo may play the same role in ecdysteroid biosynthesis in early embryos, as reported in insects. After hatching, Spo expression was repressed, while Jhamt expression was activated transiently, despite its silencing during other embryonic stages. Jhamt RNAi showed little effect on survival, but shortened the embryonic period. Exposure to the sesquiterpenoid analog Fenoxycarb extended the embryonic period and rescued the Jhamt RNAi phenotype, demonstrating a previously unidentified role of sesquiterpenoid in the repression of precocious embryogenesis. Interestingly, the knockdown of Jhamt resulted in the derepression of ecdysteroid biosynthesis genes, including Spo, similar to regulation during insect hormonal biosynthesis. Sesquiterpenoid signaling via the Methoprene-tolerant gene was found to be responsible for the repression of ecdysteroid biosynthesis genes. It upregulated an ortholog of CYP18a1 that degrades ecdysteroid in insects. These results illuminate the conserved and specific functions of the ecdysteroid and sesquiterpenoid pathways in Daphnia embryos. We also infer that the common ancestor of branchiopod crustaceans and insects exhibited antagonism between the two endocrine hormones before their divergence 400 million years ago.


Assuntos
Daphnia/genética , Ecdisteroides/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Sesquiterpenos/metabolismo , Animais , Daphnia/embriologia , Daphnia/metabolismo , Ecdisteroides/genética , Evolução Molecular , Metiltransferases/genética , Metiltransferases/metabolismo
4.
PLoS One ; 15(8): e0236633, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785249

RESUMO

The induction of general plant defense responses following the perception of external elicitors is now regarded as the first level of the plant immune response. Depending on the involvement or not of these molecules in pathogenicity, this induction of defense is called either Pathogen-Associated Molecular Pattern (PAMP) Triggered Immunity or Pattern Triggered Immunity-both abbreviated to PTI. Because PTI is assumed to be a widespread and stable form of resistance to infection, understanding the mechanisms driving it becomes a major goal for the sustainable management of plant-pathogen interactions. However, the induction of PTI is complex. Our hypotheses are that (i) the recognition by the plant of PAMPs vs non-PAMP elicitors leads to specific defense profiles and (ii) the responses specifically induced by PAMPs target critical life history traits of the pathogen that produced them. We thus analyzed, using a metabolomic approach coupled with transcriptomic and hormonal analyses, the defense profiles induced in potato foliage treated with either a Concentrated Culture Filtrate (CCF) from Phytophthora infestans or two non-PAMP preparations, ß-aminobutyric acid (BABA) and an Ulva spp. Extract, used separately. Each elicitor induced specific defense profiles. CCF up-regulated sesquiterpenes but down-regulated sterols and phenols, notably α-chaconine, caffeoyl quinic acid and rutin, which decreased spore production of P. infestans in vitro. CCF thus induces both defense and counter-defense responses. By contrast, the Ulva extract triggered the synthesis of a large-spectrum of antimicrobial compounds through the phenylpropanoid/flavonoid pathways, while BABA targeted the primary metabolism. Hence, PTI can be regarded as a heterogeneous set of general and pathogen-specific responses triggered by the molecular signatures of each elicitor, rather than as a uniform, non-specific and broad-spectrum set of general defense reactions.


Assuntos
Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Solanum tuberosum/imunologia , Aminobutiratos/farmacologia , Resistência à Doença/efeitos dos fármacos , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Fenóis/metabolismo , Phytophthora infestans/imunologia , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Sesquiterpenos/metabolismo , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/microbiologia , Esteróis/metabolismo , Ulva/química
5.
J Oleo Sci ; 69(7): 711-718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612020

RESUMO

Angelica keiskei (ashitaba) is an edible plant belonging to the Apiacea family. We focused on sesquiterpenes in the leaves eaten by humans (specifically, in the Japanese population), and confirmed the presence of several sesquiterpenes by GC-MS. Thus, total RNA was extracted from the ashitaba leaves, reverse transcribed, and the resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. Consequently, we were able to isolate two full-length Tps genes (designated AkTps1 and AkTps2). Functional analysis of these two genes was carried out with Escherichia coli cells that expressed mevalonate pathway genes to increase the substrate (farnesyl diphosphate) amount of sesquiterpene synthase, revealing that AkTps1 encodes germacrene D synthase, and AkTps2 codes for an enzyme that catalyzes the generation of germacrene B and smaller amounts of germacrene D (a germacrene B and D synthase). We proposed biosynthetic routes of these two sesquiterpenes from farnesyl diphosphate (FPP) via farnesyl cation.


Assuntos
Angelica/genética , Angelica/metabolismo , Clonagem Molecular/métodos , DNA Circular , Glucosiltransferases/isolamento & purificação , Folhas de Planta/química , Folhas de Planta/genética , RNA de Plantas/isolamento & purificação , Sesquiterpenos/análise , Sesquiterpenos/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Catálise , Escherichia coli , Cromatografia Gasosa-Espectrometria de Massas , Amplificação de Genes , Ácido Mevalônico/metabolismo , Reação em Cadeia da Polimerase , Sesquiterpenos de Germacrano/metabolismo , Transdução de Sinais/genética
6.
J Biosci Bioeng ; 130(5): 464-470, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32713813

RESUMO

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.


Assuntos
Monoterpenos Acíclicos/química , Monoterpenos Acíclicos/metabolismo , Ácido Glutâmico/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Synechocystis/metabolismo , Escherichia coli/genética , Aromatizantes/química , Aromatizantes/metabolismo , Engenharia Genética , Ácido Glutâmico/química , Estereoisomerismo , Synechocystis/genética
7.
PLoS One ; 15(7): e0235416, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614884

RESUMO

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.


Assuntos
Alquil e Aril Transferases , Proteínas de Plantas , Plectranthus/enzimologia , Monoterpenos Acíclicos/metabolismo , Alquil e Aril Transferases/biossíntese , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Clonagem Molecular , Escherichia coli/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo
8.
Nat Commun ; 11(1): 3091, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555159

RESUMO

Phytoalexins have attracted much attention due to their health-promoting effects and their vital role in plant health during the last years. Especially the 6a-hydroxypterocarpans glyceollin I and glyceollin II, which may be isolated from stressed soy plants, possess a broad spectrum of bioactivities such as anticancer activity and beneficial contributions against western diseases by anti-oxidative and anti-cholesterolemic effects. Aiming for a catalytic asymmetric access to these natural products, we establish the asymmetric syntheses of the natural isoflavonoids (-)-variabilin, (-)-homopterocarpin, (-)-medicarpin, (-)-3,9-dihydroxypterocarpan, and (-)-vestitol by means of an asymmetric transfer hydrogenation (ATH) reaction. We successfully adapt this pathway to the first catalytic asymmetric total synthesis of (-)-glyceollin I and (-)-glyceollin II. This eight-step synthesis features an efficient one-pot transformation of a 2'-hydroxyl-substituted isoflavone to a virtually enantiopure pterocarpan by means of an ATH and a regioselective benzylic oxidation under aerobic conditions to afford the susceptible 6a-hydroxypterocarpan skeleton.


Assuntos
Isoflavonas/metabolismo , Pterocarpanos/metabolismo , Sesquiterpenos/metabolismo , Produtos Biológicos/metabolismo , Biomimética/métodos , Regulação da Expressão Gênica de Plantas
9.
Plant Sci ; 294: 110463, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234219

RESUMO

German chamomile (Matricaria chamomilla L.) is one of the most ancient medicinal species in the world and terpenoids from their flowers have important medicinal value. We cloned three sesquiterpene synthase genes, McGDS1, McGDS2 and McGDS3, and performed sequence alignment and phylogenetic analysis. The encoded proteins possess three conserved structural features: an RRxxxxxxxxW motif, an RxR motif, and a DDxxD motif. McGDS1, McGDS2 and McGDS3 were confirmed to be (E)-farnesene synthase, germacrene D synthase, and germacrene A synthase, respectively. Subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus. qPCR indicated that McGDS1, McGDS2 and McGDS3, were more highly expressed in young flowers than in old flowers and the expression was highly correlated with amounts of the end-product essential oils ((E)-ß-farnesene, germacrene D and ß-elemene), with coefficients of 0.76, 0.83 and 0.68, respectively. We also established a transformation system for chamomile hairy roots. The overexpression of McGDS1, McGDS2 and McGDS3 resulted in γ-muurolene accumulation in hairy roots. The activity of three aphid alarm pheromones here forms the molecular basis for the study of the biosynthesis and regulation of volatile terpenes. Transformation of chamomile hairy roots provides a simple system in which to study terpene biosynthesis in chamomile.


Assuntos
Matricaria/metabolismo , Animais , Afídeos , Camomila/metabolismo , Flores/genética , Flores/metabolismo , Matricaria/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Reação em Cadeia da Polimerase , Sesquiterpenos/metabolismo , Sesquiterpenos de Germacrano/metabolismo
10.
Sci Rep ; 10(1): 3309, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094454

RESUMO

Cannabis research has historically focused on the most prevalent cannabinoids. However, extracts with a broad spectrum of secondary metabolites may have increased efficacy and decreased adverse effects compared to cannabinoids in isolation. Cannabis's complexity contributes to the length and breadth of its historical usage, including the individual application of the leaves, stem barks, and roots, for which modern research has not fully developed its therapeutic potential. This study is the first attempt to profile secondary metabolites groups in individual plant parts comprehensively. We profiled 14 cannabinoids, 47 terpenoids (29 monoterpenoids, 15 sesquiterpenoids, and 3 triterpenoids), 3 sterols, and 7 flavonoids in cannabis flowers, leaves, stem barks, and roots in three chemovars available. Cannabis inflorescence was characterized by cannabinoids (15.77-20.37%), terpenoids (1.28-2.14%), and flavonoids (0.07-0.14%); the leaf by cannabinoids (1.10-2.10%), terpenoids (0.13-0.28%), and flavonoids (0.34-0.44%); stem barks by sterols (0.07-0.08%) and triterpenoids (0.05-0.15%); roots by sterols (0.06-0.09%) and triterpenoids (0.13-0.24%). This comprehensive profile of bioactive compounds can form a baseline of reference values useful for research and clinical studies to understand the "entourage effect" of cannabis as a whole, and also to rediscover therapeutic potential for each part of cannabis from their traditional use by applying modern scientific methodologies.


Assuntos
Cannabis/metabolismo , Inflorescência/metabolismo , Casca de Planta/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Metabolismo Secundário , Vias Biossintéticas , Canabinoides/metabolismo , Flavonoides/metabolismo , Reprodutibilidade dos Testes , Sesquiterpenos/metabolismo , Esteróis/metabolismo
11.
Enzyme Microb Technol ; 134: 109485, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044032

RESUMO

Increasing the metabolic flux of the mevalonate pathway, reducing the metabolic flux of competing pathway and utilizing the diauxie-inducible system constructed by GAL promoters are strategies commonly used in yeast metabolic engineering for the production of terpenoids. Using these strategies, we constructed a series of yeast strains with a strengthened mevalonate pathway and finally produced 336.5 mg/L nerolidol in a shake flask. The spliced HAC1 mRNA assay indicated that the unfolded protein response (UPR) occurred in the strains that we constructed. UPR strains exhibited the low transcriptional activities of GAL1 promoter. HAC1-overexpressing strain exhibited dramatically enhanced transcriptional activity of GAL1 promoter at 72 h of fermentation in flasks. HAC1 overexpression also increased the nerolidol titer by 47.7 %, reaching 497.0 mg/L and increased cell vitality. RNA-seq showed that the genes whose transcription responded to HAC1-overexpression were involved in the regulation of monocarboxylic acid metabolic processes and cellular amino acid biosynthetic process, indicating that the metabolic regulation may be part of the reason of the improved nerolidol synthesis. Our findings enrich the knowledge of the relationship between the construction of sesquiterpene-producing cell factories and UPR regulation. This study provides an effective strategy for sesquiterpene production in yeast.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos/metabolismo , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Engenharia Metabólica , Regiões Promotoras Genéticas , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
12.
J Agric Food Chem ; 68(10): 3214-3219, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32079394

RESUMO

Four terpene synthases for the biosynthesis of volatile terpenoids were identified from the transcriptome of Stellera chamaejasme L. flowers, including SchTPS1, SchTPS2, SchTPS3, and SchTPS4. Their functions were characterized by synthetic biology approaches in Escherichia coli and in vitro enzymatic assays. SchTPS1, SchTPS2, and SchTPS3 are guaiene synthases, while SchTPS4 is an (E,E)-geranyl linalool synthase. Next, SchTPS1 and α-guaiene 2-oxidase VvSTO2 were co-expressed in Saccharomyces cerevisiae to reconstruct the biosynthetic pathway of (-)-rotundone, which is a unique aroma compound in fruits, vegetables, and wines. This is the first report for the construction of a (-)-rotundone-producing microbial platform.


Assuntos
Alquil e Aril Transferases/metabolismo , Azulenos/metabolismo , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Sesquiterpenos de Guaiano/metabolismo , Sesquiterpenos/metabolismo , Thymelaeaceae/enzimologia , Alquil e Aril Transferases/genética , Vias Biossintéticas , Flores/enzimologia , Flores/genética , Expressão Gênica , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Thymelaeaceae/genética
13.
Sci Rep ; 10(1): 141, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924820

RESUMO

1ß-hydroxy alantolactone, a sesquiterpene lactone, exhibits potent anti-inflammatory and anticancer activities. Recently, it has been found to target UbcH5s by covalently bonding with Cys85 specifically, but the exact molecular basis remains unclear. Here, we analyzed the structural specificity of the catalytic site of UbcH5s by comparing them with other E2 proteins. Molecular dynamics was performed to detect the structural stability of the catalytic site. Docking method was then used to predict conformations of ligand docked at the catalytic site of UbcH5s. The electrostatic surface and charge distribution of ligand and proteins were analyzed by quantitative calculation. Molecular dynamics was used to detect the stability of docking complexes of 1ß-hydroxy alantolactone and UbcH5s, the covalently bonded intermediates and the products. The QM/MM methodology was used to calculate the free energy barrier of hydrogen transfer and formation of covalent bond between 15-position carbon of ligand and Cys85. Results revealed that the structure of the catalytic site is stable, and 1ß-hydroxy alantolactone can dock at the catalytic site with correct conformation. Molecular dynamics further demonstrates that 1ß-hydroxy alantolactone can steadily combine with UbcH5s. Intermediate and product of catalytic reaction are also certified to be stable. Besides, Asp112 and Asn114 function as anchors to fix ligand, ensuring it steadily docked at catalytic site to complete covalent reaction. More importantly, we have found that Cys85 of UbcH5c is more efficient to form a covalent bond with the ligand in comparison with UbcH5a and UbcH5b. Our results successfully explained the mechanism of 1ß-hydroxy alantolactone covalently bonding with UbcH5s. Such molecular mechanism may provide a better insight into the molecular development or modification for ubiquitin-related drugs.


Assuntos
Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Sesquiterpenos/farmacologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Sequência de Aminoácidos , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Humanos , Alinhamento de Sequência , Sesquiterpenos/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
14.
Gen Comp Endocrinol ; 289: 113394, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962126

RESUMO

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.


Assuntos
Cromatografia Líquida/métodos , Heterópteros/química , Insetos/química , Sesquiterpenos/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Estereoisomerismo
15.
J Agric Food Chem ; 68(5): 1382-1389, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31944688

RESUMO

Metabolic engineering of Saccharomyces cerevisiae focusing on the cytoplasm for sustainable terpenoid production is commonly practiced. However, engineering organelles for terpenoid production is rarely reported. Herein, peroxisomes, together with the cytoplasm, were engineered to boost sesquiterpene α-humulene synthesis in S. cerevisiae. The farnesyl diphosphate synthetic pathway and α-humulene synthase were successfully expressed inside yeast peroxisomes to enable high-level α-humulene production with glucose as the sole carbon source. With the combination of peroxisomal and cytoplasmic engineering, α-humulene production was increased by 2.5-fold compared to that in cytoplasm-engineered recombinant strains. Finally, the α-humulene titer of 1726.78 mg/L was achieved by fed-batch fermentation in a 5 L bioreactor. The strategy presented here offers an efficient method for terpenoid production in S. cerevisiae.


Assuntos
Acetilcoenzima A/metabolismo , Citosol/metabolismo , Sesquiterpenos Monocíclicos/metabolismo , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Engenharia Metabólica , Peroxissomos/genética , Sesquiterpenos/metabolismo
16.
Xenobiotica ; 50(3): 354-362, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31132291

RESUMO

1. More than 30% of epilepsy patients suffer pharmacoresistance. Transport of antileptic drugs by P-glycoprotein (P-gp) and MRP2 plays an important role in drug-resistant epilepsy. Huperzine A (Hup-A) is a natural compound, which might have potential in treating neurological disorders including epilepsy and Alzheimer's disease. In this study, we investigated whether human P-gp and MRP2 transport Hup-A.2. LLC-PK1 and MDCKII cells transfected with human P-gp or MRP2 were used to establish concentration equilibrium transport assays (CETAs) and determine the transport profile of Hup-A. The expression of P-gp and MRP2 was detected by qPCR and western blotting. The transport function of P-gp and MRP2 was measured by Rho123 and CDFDA cell uptake assay.3. In CETAs, Hup-A at concentrations of 10 ng/mL or 2 µg/mL was transported by MDR1 and MRP2 from basolateral to apical sides of the cell monolayers. P-gp and MRP2 inhibitors completely blocked the efflux of Hup-A. There was no efflux of Hup-A in LLC-PK1 or MDCKII wild-type (WT) cells.4. We demonstrate that Hup-A is a substrate of P-gp and MRP2. These results imply the efflux of Hup-A across the blood-brain barrier (BBB) in vivo, suggesting potential drug resistance of Hup-A.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Alcaloides/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Sesquiterpenos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Transporte Biológico , Barreira Hematoencefálica/metabolismo
17.
Plant Cell Environ ; 43(1): 209-222, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31702837

RESUMO

Redox processes regulate plant/insect responses, but the precise roles of environmental triggers and specific molecular components remain poorly defined. Aphid fecundity and plant responses were therefore measured in Arabidopsis thaliana mutants deficient in either catalase 2 (cat2), different protein phosphatase 2A (PP2A) subunits or glutathione (cad2, pad2, and clt1) under either moderate (250 µmol m-2 s-1 ) or high (800 µmol m-2 s-1 ) light. Aphid fecundity was decreased in pp2a-b'γ, cat2 and the cat2 pp2a-b'γ double mutants relative to the wild type under moderate irradiance. High light decreased aphid numbers in all genotypes except for cat2. Aphid fecundity was similar in the cat2 and glutathione-, phytoalexin-, and glucosinolate-deficient cat2cad2 double mutants under both irradiances. Aphid-induced increases in transcripts encoding the abscisic acid-related ARABIDOPSIS ZINC-FINGER PROTEIN 1 transcription factor were observed only under moderate light. Conversely, aphid induced increases in transcripts encoding the jasmonate-synthesis enzyme ALLENE OXIDE CYCLASE 3 was observed in all genotypes only under high light. Aphid-induced increases in REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 mRNAs were observed in all genotypes except pp2a-b'ζ1-1 under both irradiances. Aphid fecundity is therefore regulated by cellular redox signalling that is mediated, at least in part, through PP2A-dependent mitochondria to nucleus signalling pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Catalase/metabolismo , Glutationa/metabolismo , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/fisiologia , Animais , Afídeos/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catalase/genética , Proteínas de Transporte de Cobre/metabolismo , Regulação da Expressão Gênica de Plantas , Glucosinolatos/metabolismo , Glutationa/genética , Luz , Oxirredução , Folhas de Planta/metabolismo , Proteína Fosfatase 2/genética , RNA Mensageiro , Sesquiterpenos/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma
18.
Plant Cell Rep ; 39(2): 207-215, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31713663

RESUMO

KEY MESSAGE: Benzoate-Coenzyme A ligase enzyme activity catalyzing the conversion of free benzoic acid to benzoyl-CoA was detected and biochemically characterized in the elicitor-treated pear cell cultures. Asian pear (Pyrus pyrifolia) is an economically and nutritionally important fruit-bearing tree of the subtribe Malinae. Upon pathogen attack, pears produce unique benzoate-derived biphenyl phytoalexins. The upstream biosynthesis of the biphenyl in Malinae is still incomplete. Previously, protein preparations from yeast extract-treated pear cultures were able to convert L-phenylalanine to cinnamic acid catalyzed by the activity of the phenylalanine ammonia lyase. The same extract was able to perform a C2 side-chain cleavage of cinnamic acid to benzaldehyde followed by oxidation of the latter to benzoic acid owing to the molecularly-undefined benzaldehyde synthase and benzaldehyde dehydrogenase activities, respectively. The biosynthesis of biphenyls starts with benzoate-Coenzyme A ligase (BZL), which converts benzoic acid to benzoyl-CoA. Subsequently, the previously-defined biphenyl synthase uses benzoyl-CoA to form the biphenyls. The current study reports the first time detection and characterization of BZL activity in elicitor-treated pear cell cultures. The preferred substrate was benzoic acid (Km = 62 ± 4 µM). Magnesium or manganese was prerequisite for the activity, which was enhanced by ~ 70% in the presence of potassium. Maximum BZL activity was observed 18 h post elicitation, which is in agreement with the coordinate induction reported for the enzymes in the same pathway. The induced BZL activity preceded the accumulation of biphenyls supporting its involvement in their biosynthesis.


Assuntos
Compostos de Bifenilo/metabolismo , Coenzima A Ligases/genética , Células Vegetais , Pyrus/citologia , Sesquiterpenos/metabolismo , Acil Coenzima A/metabolismo , Benzaldeídos/metabolismo , Ácido Benzoico/metabolismo , Cinamatos/metabolismo , Coenzima A Ligases/metabolismo , Fenilalanina Amônia-Liase/metabolismo , Pyrus/metabolismo , Espectrometria de Massas em Tandem
19.
J Nat Med ; 74(1): 98-105, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31392566

RESUMO

Agarwood, which is used as medicine and incense, contains sesquiterpenes and chromones. Agarotetrol is a chromone derivative found in high concentrations in the water-extract fraction of agarwood and thus may be present in pharmaceutical products made from decoctions of agarwood. Agarotetrol has been reported to be present at the early stages of cell death in calli. We therefore examined the presence of agarotetrol in medical- and incense-grade agarwood, in agarwood-source plants lacking resin deposits, and in artificially made agarwood. Agarotetrol appeared as a large peak in the HPLC chromatograms of all samples of medical-grade and artificially made agarwood, and in most incense-grade agarwood samples. In contrast, agarwood samples lacking resin deposits did not contain agarotetrol. These results show that agarotetrol is characteristic of resin formation. Agarotetrol was also detected in decoctions of agarwood. A newly developed TLC method for the detection of agarotetrol in agarwood is described.


Assuntos
Cromonas/análise , Cromonas/química , Resinas Vegetais/análise , Thymelaeaceae/química , Cromatografia Líquida de Alta Pressão , Extratos Vegetais , Resinas Vegetais/química , Sesquiterpenos/metabolismo
20.
J Chem Ecol ; 46(1): 63-75, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31832894

RESUMO

Plants experience seasonal fluctuations in abiotic and biotic factors such as herbivore attack rates. If and how root defense expression co-varies with seasonal fluctuations in abiotic factors and root herbivore attack rates is not well understood. Here, we evaluated seasonal changes in defensive root latex chemistry of Taraxacum officinale plants in the field and correlated the changes with seasonal fluctuations in abiotic factors and damage potential by Melolontha melolontha, a major natural enemy of T. officinale. We then explored the causality and consequences of these relationships under controlled conditions. The concentration of the defensive sesquiterpene lactone taraxinic acid ß-D glucopyranosyl ester (TA-G) varied substantially over the year and was most strongly correlated to mean monthly temperature. Both temperature and TA-G levels were correlated with annual fluctuations in potential M. melolontha damage. Under controlled conditions, plants grown under high temperature produced more TA-G and were less attractive for M. melolontha. However, temperature-dependent M. melolontha feeding preferences were not significantly altered in TA-G deficient transgenic lines. Our results suggest that fluctuations in temperature leads to variation in the production of a root defensive metabolites that co-varies with expected attack of a major root herbivore. Temperature-dependent herbivore preference, however, is likely to be modulated by other phenotypic alterations.


Assuntos
Besouros/fisiologia , Glucosídeos/metabolismo , Lactonas/metabolismo , Sesquiterpenos/metabolismo , Taraxacum/química , Animais , Biomassa , Besouros/crescimento & desenvolvimento , Glucosídeos/química , Glucosídeos/farmacologia , Herbivoria/efeitos dos fármacos , Lactonas/química , Lactonas/farmacologia , Larva/efeitos dos fármacos , Larva/fisiologia , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Raízes de Plantas/parasitologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Estações do Ano , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Taraxacum/metabolismo , Taraxacum/parasitologia , Temperatura
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