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1.
J Agric Food Chem ; 67(38): 10624-10636, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31483633

RESUMO

The freshness and color quality of postharvest tea leaves can be markedly prolonged and retained by proper preservation measures. Here, we investigated the dynamic changes of chlorophyll and its derivatives in postharvest tea leaves under different low-temperature treatments using natural withering as a control. Chlorophyll decomposition was found closely related with chlorophyllide, pheophorbide, and pheophytin. Low-temperature withering could slow chlorophyll degradation in postharvest tea leaves via significant inhibition on the enzyme activity and gene expression of Mg-dechelatase, chlorophyllase, and pheophorbide a oxygenase. At the initial stage of withering, a significant increase was observed in the chlorophyll content, expression of chlorophyll-synthesis-related enzymes (such as glutamyl-tRNA synthetase, etc.), and chlorophyll synthase activity in newly picked tea leaves. Moreover, an obvious decrease was found in the content of l-glutamate as the foremost precursor substance of chlorophyll synthesis. Hence, our findings revealed that the chlorophyll synthesis reaction was induced by the light-dehydration-stress in the initial withering of tea leaves. This study provides a theoretical basis for exploring preservation technology in actual green tea production.


Assuntos
Camellia sinensis/genética , Camellia sinensis/metabolismo , Clorofila/metabolismo , Manipulação de Alimentos/métodos , Regulação da Expressão Gênica de Plantas , Camellia sinensis/química , Camellia sinensis/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Clorofila/química , Cor , Enzimas/genética , Enzimas/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura Ambiente
3.
Nat Commun ; 10(1): 2043, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053724

RESUMO

Unlike proteins, glycan chains are not directly encoded by DNA, but by the specificity of the enzymes that assemble them. Theoretical calculations have proposed an astronomical number of possible isomers (> 1012 hexasaccharides) but the actual diversity of glycan structures in nature is not known. Bacteria of the Bacteroidetes phylum are considered primary degraders of polysaccharides and they are found in all ecosystems investigated. In Bacteroidetes genomes, carbohydrate-degrading enzymes (CAZymes) are arranged in gene clusters termed polysaccharide utilization loci (PULs). The depolymerization of a given complex glycan by Bacteroidetes PULs requires bespoke enzymes; conversely, the enzyme composition in PULs can provide information on the structure of the targeted glycans. Here we group the 13,537 PULs encoded by 964 Bacteroidetes genomes according to their CAZyme composition. We find that collectively Bacteroidetes have elaborated a few thousand enzyme combinations for glycan breakdown, suggesting a global estimate of diversity of glycan structures much smaller than the theoretical one.


Assuntos
Proteínas de Bactérias/genética , Bacteroidetes/genética , Enzimas/genética , Genoma Bacteriano/genética , Polissacarídeos/metabolismo , Proteínas de Bactérias/metabolismo , Bacteroidetes/metabolismo , Enzimas/metabolismo , Loci Gênicos , Isomerismo , Polissacarídeos/química
4.
Nat Commun ; 10(1): 2142, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086174

RESUMO

Metabolic engineers endeavor to create a bio-based manufacturing industry using microbes to produce fuels, chemicals, and medicines. Plant natural products (PNPs) are historically challenging to produce and are ubiquitous in medicines, flavors, and fragrances. Engineering PNP pathways into new hosts requires finding or modifying a suitable host to accommodate the pathway, planning and implementing a biosynthetic route to the compound, and discovering or engineering enzymes for missing steps. In this review, we describe recent developments in metabolic engineering at the level of host, pathway, and enzyme, and discuss how the field is approaching ever more complex biosynthetic opportunities.


Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/metabolismo , Plantas/metabolismo , Vias Biossintéticas/genética , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/tendências , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Biologia Sintética/tendências
5.
Appl Microbiol Biotechnol ; 103(10): 4177-4192, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30968165

RESUMO

Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.


Assuntos
Biotransformação , Enzimas/genética , Marcadores Genéticos , Consórcios Microbianos/genética , Xilenos/metabolismo , Anaerobiose , Metagenômica
6.
Phytomedicine ; 59: 152901, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30981189

RESUMO

BACKGROUND: Diabetes mellitus poses serious threat to the global population due to the alarming diabetic complications it leads to. The current therapeutic options available can be improved for better efficiency and maximum benefits. Combination therapy has been commonly used to improve the efficacy and to minimize the side effects of drugs in current clinical use. PURPOSE: The present study aims to assess the interaction between a natural molecule mangiferin with the commercially available oral hypoglycemic drugs metformin and gliclazide in diabetic rats. METHODS: In this study, the in vitro cytotoxicity and glucose uptake studies were performed in HepG2 cells. Based on experimental data, the combination index of the hypoglycemic drugs like metformin and gliclazide in combination with different doses of mangiferin was determined using COMPUSYN software. Further, in vivo studies were performed in HFD + STZ induced diabetic male Sprague Dawley rats. Serum parameters, enzyme markers, hepatic oxidative stress markers, gene and protein expression studies and histopathological analyses were performed in rat liver to identify the mode of action of the combination drug administration. RESULTS: The in vitro studies on HepG2 cells suggest a positive interaction of mangiferin with both metformin and gliclazide at specific concentrations as evidenced by glucose uptake. The hepatic enzymes, oxidative stress markers, carbohydrate metabolizing enzymes, gene (AMPK, Akt, ACC ß and Glut-2) and protein (PPARα, PPARγ) expression confirmed the results of the in vitro studies. Both the combinations of mangiferin with metformin and mangiferin with gliclazide exhibited potent antidiabetic effect. The combination of mangiferin with metformin was insulin dependent (Akt pathway) whereas the combination of mangiferin and gliclazide was insulin independent (AMPK pathway). CONCLUSION: The overall results suggest that combination of mangiferin with both metformin and gliclazide alleviates diabetic conditions potentially at specific doses and modulates the adverse effect of high dose of commonly used OHD's. This combination therapy can be translated for its clinical use as a diabetes management strategy.


Assuntos
Gliclazida/farmacologia , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Xantonas/farmacologia , Administração Oral , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Quimioterapia Combinada , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Gliclazida/administração & dosagem , Células Hep G2 , Humanos , Insulina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metformina/administração & dosagem , Ratos Sprague-Dawley
7.
Org Lett ; 21(9): 3148-3152, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30990701

RESUMO

Feeding studies indicate a possible synthetic pattern for the N-terminal cis-aminocyclopentane carboxylic acid (ACPC) and suggest an unusual source of the high-carbon sugar skeleton of amipurimycin (APM). The biosynthetic gene cluster of APM was identified and confirmed by in vivo experiments. A C9 core intermediate was discovered from null mutants of ACPC pathway, and an ATP-grasp enzyme (ApmA8) was reconstituted in vitro for ACPC loading. Our observations allow a first proposal of the APM biosynthetic pathway.


Assuntos
Antibacterianos/biossíntese , Família Multigênica , Nucleosídeos/biossíntese , Purinas/biossíntese , Açúcares/química , Trifosfato de Adenosina/metabolismo , Antibacterianos/química , Vias Biossintéticas/genética , Cicloleucina/química , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Nucleosídeos/química , Purinas/química , Streptomyces/genética , Streptomyces/metabolismo
8.
J Microbiol Biotechnol ; 29(4): 625-632, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30954032

RESUMO

The unified saccharification and fermentation (USF) system was developed for direct production of ethanol from agarose. This system contains an enzymatic saccharification process that uses three types of agarases and a fermentation process by recombinant yeast. The pGMFα-HGN plasmid harboring AGAH71 and AGAG1 genes encoding ß-agarase and the NABH558 gene encoding neoagarobiose hydrolase was constructed and transformed into the Saccharomyces cerevisiae 2805 strain. Three secretory agarases were produced by introducing an S. cerevisiae signal sequence, and they efficiently degraded agarose to galactose, 3,6-anhydro- L-galactose (AHG), neoagarobiose, and neoagarohexose. To directly produce ethanol from agarose, the S. cerevisiae 2805/pGMFα-HGN strain was cultivated into YP-containing agarose medium at 40°C for 48 h (for saccharification) and then 30°C for 72 h (for fermentation). During the united cultivation process for 120 h, a maximum of 1.97 g/l ethanol from 10 g/l agarose was produced. This is the first report on a single process containing enzymatic saccharification and fermentation for direct production of ethanol without chemical liquefaction (pretreatment) of agarose.


Assuntos
Etanol/metabolismo , Fermentação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sefarose/metabolismo , Meios de Cultura , Dissacaridases/genética , Dissacarídeos/metabolismo , Enzimas/genética , Escherichia coli/genética , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Glicosídeo Hidrolases/genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo
9.
Phytomedicine ; 58: 152874, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30889421

RESUMO

BACKGROUND: The fruits of Psoralea corylifolia L. (Fructus Psoraleae, FP) has a long history and a wide range of applications in the treatment of osteoporosis and leukoderma. Although it is well known that FP could cause hepatotoxicity and reproductive toxicity, less is known about its potential toxicity on multiple organs. PURPOSE: This study aims to determine the multiorgan toxicity of EtOH extract of FP (EEFP) and to investigate the underlying mechanisms through a systematic evaluation in Wistar rats. STUDY DESIGN AND METHODS: Wistar rats were orally administered with the EEFP at doses of 1.5, 1.0 and 0.5 g/kg for 28 days. Histopathologic and clinicopathologic analyses were performed, and the hormone levels in serum and the mRNA levels of enzymes related to the production of steroid hormones in adrenal glands were detected. The area of each band of adrenal glands and the steroid levels in the adrenal glands were also measured. RESULTS: After the treatment, both the histopathologic and clinicopathologic examination showed that EEFP caused liver, prostate, seminal vesicle and adrenal gland damage. Among the enzymes involved in the regulation of adrenal steroid hormone production, NET, VMAT2, and CYP11B1 were upregulated, while CYP17A1 was downregulated. Among the adrenal steroid hormones, COR and NE were upregulated, while levels of DHT and serum ACRH and CRH decreased. CONCLUSION: Our results indicated that adrenal gland, prostate, and seminal vesicles could also be the target organs of FP-induced toxicity. Abnormal enzyme and hormone production related to the hypothalamic pituitary adrenal (HPA) axis caused by the EEFP may be the potential toxic mechanism for changes in the adrenal gland and secondary sex organs of male rats.


Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Enzimas/metabolismo , Extratos Vegetais/toxicidade , Esteroides/metabolismo , Administração Oral , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Animais , Enzimas/genética , Etanol/química , Feminino , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Próstata/efeitos dos fármacos , Próstata/patologia , Ratos Wistar , Testes de Toxicidade/métodos
10.
Int J Biol Macromol ; 130: 186-196, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817963

RESUMO

Enzymes as green industrial biocatalysts have become a powerful norm that offers several advantages over traditional catalytic agents with regard to process efficiency, reusability, sustainability, and overall cost-effective ratio. However, enzymes obtained from natural origins are often engineered/tailored since their native forms do not fulfill the acute need for the industrial application. Revolutionary developments in protein engineering provide excellent opportunities for designing and constructing novel industrial biocatalysts with improved functional properties including catalytic activity, stability, substrate specificity, and reaction product inhibition. Momentum in enzyme immobilization has enabled robustness and optimal functions in extreme industrial environments, such as high temperature or organic solvents. The emergence of multi-enzyme catalytic cascade based on a combination of biocatalysts presents multifarious opportunities in biosynthesis, biocatalysis, and biotransformation. This review focuses on the emerging and state-of-the-art enzyme engineering trends and approaches to constructing innovative nano- and microstructured biocatalysts with enhanced catalytic activity and stability features requisite for industrial exploitation. Continuous key developments in this direction together with protein engineering in unique ways might offer ever-increasing opportunities for future biocatalysis-based industrial bioprocesses.


Assuntos
Enzimas/metabolismo , Biocatálise , Evolução Molecular Direcionada , Engenharia , Enzimas/química , Enzimas/genética , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/metabolismo , Modelos Moleculares
11.
Nature ; 568(7750): 122-126, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30867595

RESUMO

Pericyclic reactions are powerful transformations for the construction of carbon-carbon and carbon-heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest1. [4+2] cycloadditions (Diels-Alder reactions) have been widely used in organic synthesis2 for the formation of six-membered rings and are now well-established in biosynthesis3-6. [6+4] and other 'higher-order' cycloadditions were predicted7 in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system8,9. However, although enzyme-catalysed [6+4] cycloadditions have been proposed10-12, they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals11,12. The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states.


Assuntos
Biocatálise , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Reação de Cicloadição , Enzimas/metabolismo , Lactonas/química , Lactonas/metabolismo , Cristalografia por Raios X , Teoria da Densidade Funcional , Enzimas/química , Enzimas/genética , Simulação de Dinâmica Molecular , Conformação Proteica , Termodinâmica
12.
Proc Natl Acad Sci U S A ; 116(13): 6063-6068, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30850540

RESUMO

Over the last two decades, the number of gene/protein sequences gleaned from sequencing projects of individual genomes and environmental DNA has grown exponentially. Only a tiny fraction of these predicted proteins has been experimentally characterized, and the function of most proteins remains hypothetical or only predicted based on sequence similarity. Despite the development of postgenomic methods, such as transcriptomics, proteomics, and metabolomics, the assignment of function to protein sequences remains one of the main challenges in modern biology. As in all classes of proteins, the growing number of predicted carbohydrate-active enzymes (CAZymes) has not been accompanied by a systematic and accurate attribution of function. Taking advantage of the CAZy database, which groups CAZymes into families and subfamilies based on amino acid similarities, we recombinantly produced 564 proteins selected from subfamilies without any biochemically characterized representatives, from distant relatives of characterized enzymes and from nonclassified proteins that show little similarity with known CAZymes. Screening these proteins for activity on a wide collection of carbohydrate substrates led to the discovery of 13 CAZyme families (two of which were also discovered by others during the course of our work), revealed three previously unknown substrate specificities, and assigned a function to 25 subfamilies.


Assuntos
Metabolismo dos Carboidratos , Enzimas/genética , Análise de Sequência de Proteína , Sequência de Aminoácidos , Animais , Metabolismo dos Carboidratos/genética , Enzimas/metabolismo , Genômica/métodos , Humanos , Polissacarídeos/metabolismo , Análise de Sequência de DNA , Relação Estrutura-Atividade
13.
Proc Natl Acad Sci U S A ; 116(15): 7298-7307, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30910961

RESUMO

Thousands of biochemical reactions with characterized activities are "orphan," meaning they cannot be assigned to a specific enzyme, leaving gaps in metabolic pathways. Novel reactions predicted by pathway-generation tools also lack associated sequences, limiting protein engineering applications. Associating orphan and novel reactions with known biochemistry and suggesting enzymes to catalyze them is a daunting problem. We propose the method BridgIT to identify candidate genes and catalyzing proteins for these reactions. This method introduces information about the enzyme binding pocket into reaction-similarity comparisons. BridgIT assesses the similarity of two reactions, one orphan and one well-characterized nonorphan reaction, using their substrate reactive sites, their surrounding structures, and the structures of the generated products to suggest enzymes that catalyze the most-similar nonorphan reactions as candidates for also catalyzing the orphan ones. We performed two large-scale validation studies to test BridgIT predictions against experimental biochemical evidence. For the 234 orphan reactions from the Kyoto Encyclopedia of Genes and Genomes (KEGG) 2011 (a comprehensive enzymatic-reaction database) that became nonorphan in KEGG 2018, BridgIT predicted the exact or a highly related enzyme for 211 of them. Moreover, for 334 of 379 novel reactions in 2014 that were later cataloged in KEGG 2018, BridgIT predicted the exact or highly similar enzymes. BridgIT requires knowledge about only four connecting bonds around the atoms of the reactive sites to correctly annotate proteins for 93% of analyzed enzymatic reactions. Increasing to seven connecting bonds allowed for the accurate identification of a sequence for nearly all known enzymatic reactions.


Assuntos
Bases de Dados de Proteínas , Enzimas , Anotação de Sequência Molecular , Análise de Sequência de Proteína , Sítios de Ligação , Enzimas/química , Enzimas/genética
14.
Microbiologyopen ; 8(4): e00572, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30851083

RESUMO

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Enzimas/genética , Biblioteca Gênica , Metagenômica/métodos , Solo/química , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Enzimas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Microbiologia do Solo
15.
Bioresour Technol ; 279: 350-361, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30755321

RESUMO

Thermozymes (from thermophiles or hyperthermophiles) offer obvious advantages due to their excellent thermostability, broad pH adaptation, and hydrolysis ability, resulting in diverse industrial applications including food, paper, and textile processing, biofuel production. However, natural thermozymes with low yield and poor adaptability severely hinder their large-scale applications. Extensive studies demonstrated that using genetic modifications such as directed evolution, semi-rational design, and rational design, expression regulations and chemical modifications effectively improved enzyme's yield, thermostability and catalytic efficiency. However, mechanism-based techniques for thermozymes improvements and applications need more attention. In this review, stabilizing mechanisms of thermozymes are summarized for thermozymes improvements, and these improved thermozymes eventually have large-scale industrial applications.


Assuntos
Enzimas/metabolismo , Archaea/metabolismo , Biocombustíveis , Estabilidade Enzimática , Enzimas/genética , Temperatura Alta , Humanos , Hidrólise
17.
Gen Comp Endocrinol ; 275: 51-64, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30721659

RESUMO

Temperature has unequivocal effects on several aspects of fish physiology, but the full extent of its interaction with key endocrine signaling systems to influence metabolic function remains unknown. The aim of the current study was to assess the individual and combined effects of elevated temperature and hyperthyroidism on hepatic metabolism in juvenile lake whitefish by quantifying mRNA abundance and activity of key metabolic enzymes. Fish were exposed to 13 (control), 17 or 21 °C for 0, 4, 8 or 24 days in the presence or absence of low-T4 (1 µg × g body weight-1) or high-T4 (10 µg × g body weight-1) treatment. Our results demonstrate moderate sensitivity to elevated temperature in this species, characterized by short-term changes in mRNA abundance of several metabolic enzymes and long-term declines in citrate synthase (CS) and cytochrome c oxidase (COX) activities. T4-induced hyperthyroidism also had several short-term effects on mRNA abundance of metabolic transcripts, including depressions in acetyl-coA carboxylase ß (accß) and carnitine palmitoyltransferase 1ß (cpt1ß), and stabilization of cs mRNA levels; however, these effects were primarily limited to elevated temperature groups, indicating temperature-dependent effects of exogenous T4 treatment in this species. In contrast, maximal CS and COX activities were not altered by hyperthyroidism at any temperature. Collectively, our data suggest that temperature has the potential to manipulate thyroid hormone physiology in juvenile lake whitefish and, under warm-conditions, hyperthyroidism may suppress certain elements of the ß-oxidation pathway without substantial impacts on overall cellular oxidative capacity.


Assuntos
Metabolismo Energético , Enzimas/genética , Enzimas/metabolismo , Metabolismo dos Lipídeos , Salmonidae , Temperatura Ambiente , Tiroxina/farmacologia , Animais , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Embrião não Mamífero , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Lagos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Oxirredução/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Salmonidae/embriologia , Salmonidae/genética , Salmonidae/metabolismo
18.
Plant Physiol Biochem ; 137: 14-24, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30710795

RESUMO

Glutamyl-tRNA reductase1 (HEMA1) and ferrochelatase1 (FC1) are both expressed in response to salt stress in the biosynthetic pathway of tetrapyrroles. Peanut (Arachis hypogaea L.) HEMA1 and FC1 were isolated by RT-PCR. The amino acid sequence encoded by the two genes showed high similarity with that in other plant species. The AhFC1 fusion protein was verified to function in chloroplast using Arabidopsis mesophyll protoplast. Sense and wild-type (WT) tobaccos were used to further study the physiological effects of AhHEMA1 and AhFC1. Compared with WT, the Heme contents and germination rate were higher in AhFC1 overexpressing plants under salt stress. Meanwhile, overexpressing AhHEMA1 also led to higher ALA and chlorophyll contents and multiple physiological changes under salt stress, such as higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), lower contents of reactive oxygen species (ROS) and slighter membrane damage. In addition, the activities of CAT, POD and APX in the AhFC1 overexpressing plants were significantly higher than that in WT lines under salt stress, but the activity of SOD between the WT plants and the transgenic plants did not exhibit significant differences. These results suggested that, peanut can enhance resistance to salt stress by improving the biosynthesis of tetrapyrrole biosynthetic.


Assuntos
Arachis/genética , Proteínas de Plantas/genética , Estresse Salino/genética , Tabaco/genética , Ácido Aminolevulínico/metabolismo , Membrana Celular/metabolismo , Clorofila/genética , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Enzimas/genética , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Heme/biossíntese , Heme/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino/fisiologia , Plântula/genética , Plântula/metabolismo , Tetrapirróis/genética , Tetrapirróis/metabolismo , Tabaco/fisiologia
19.
Methods Mol Biol ; 1923: 113-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30737737

RESUMO

Yeasts, like Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Yarrowia lipolytica, and Saccharomyces cerevisiae are frequently used producers of recombinant enzymes, particularly when posttranslational modifications are mandatory to obtain full functionality. The wide-range transformation/expression platform presented in this chapter can be used to select the optimal yeast host for high-level synthesis of the desired enzyme with favorable biochemical properties. This platform is composed of a selection marker and up to four expression modules in a linearized cassette. Here we describe the protocols for the assembly as well as the transformation of yeast strains with the respective cassettes, screening of transformants, the isolation and biochemical characterization of the enzymes, and finally a simple fermentation strategy to achieve maximal yields of the chosen recombinant enzyme.


Assuntos
Enzimas/metabolismo , Biologia Molecular/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Leveduras/genética , Enzimas/química , Enzimas/genética , Fermentação , Vetores Genéticos , Microrganismos Geneticamente Modificados , Plasmídeos/genética , Reação em Cadeia da Polimerase , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Transformação Genética , Leveduras/metabolismo
20.
Nucleic Acids Res ; 47(5): 2143-2159, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30698754

RESUMO

tRNA are post-transcriptionally modified by chemical modifications that affect all aspects of tRNA biology. An increasing number of mutations underlying human genetic diseases map to genes encoding for tRNA modification enzymes. However, our knowledge on human tRNA-modification genes remains fragmentary and the most comprehensive RNA modification database currently contains information on approximately 20% of human cytosolic tRNAs, primarily based on biochemical studies. Recent high-throughput methods such as DM-tRNA-seq now allow annotation of a majority of tRNAs for six specific base modifications. Furthermore, we identified large gaps in knowledge when we predicted all cytosolic and mitochondrial human tRNA modification genes. Only 48% of the candidate cytosolic tRNA modification enzymes have been experimentally validated in mammals (either directly or in a heterologous system). Approximately 23% of the modification genes (cytosolic and mitochondrial combined) remain unknown. We discuss these 'unidentified enzymes' cases in detail and propose candidates whenever possible. Finally, tissue-specific expression analysis shows that modification genes are highly expressed in proliferative tissues like testis and transformed cells, but scarcely in differentiated tissues, with the exception of the cerebellum. Our work provides a comprehensive up to date compilation of human tRNA modifications and their enzymes that can be used as a resource for further studies.


Assuntos
Enzimas/análise , Enzimas/genética , RNA de Transferência/metabolismo , Citosol/metabolismo , Humanos , Especificidade de Órgãos/genética , Proteômica , RNA de Transferência/química , RNA de Transferência/genética
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