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1.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
2.
Ecotoxicol Environ Saf ; 204: 111060, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32768747

RESUMO

OBJECTIVE: Serratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. METHODS: Using Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. RESULTS: ChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). CONCLUSION: ChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.


Assuntos
Proteínas de Bactérias/genética , Compostos de Cromo/metabolismo , Poluentes Ambientais/metabolismo , Genes Bacterianos , Proteínas de Membrana/genética , Microrganismos Geneticamente Modificados/genética , Serratia/genética , Biodegradação Ambiental , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Modelos Teóricos , Oxirredução , Oxirredutases/metabolismo , Serratia/metabolismo
3.
PLoS One ; 15(8): e0237577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790741

RESUMO

Abnormal skin melanin homeostasis results in refractory pigmentary diseases. Melanogenesis is influenced by gene regulation, ultraviolet radiation, and host epigenetic responses. Steroid receptor RNA activator (SRA), a long noncoding RNA, is known to regulate steroidogenesis and tumorigenesis. However, how SRA contributes to melanogenesis remains unknown. Using RNA interference against SRA in B16 and A375 melanoma cells, we observed increased pigmentation and increased expression of TRP1 and TRP2 at transcriptional and translational levels only in B16 cells. The constitutive phosphorylation of p38 in B16-shCtrl cells was inhibited in cells with knocked down SRAi. Moreover, the melanin content of control B16 cells was increased by SB202190, a p38 inhibitor. Furthermore, reduced p38 phosphorylation, enhanced TRP1 expression, and hypermelanosis were observed in A375 cells with RNA interference. These results indicate that SRA-p38-TRP1 axis has a regulatory role in melanin homeostasis and that SRA might be a potential therapeutic target for treating pigmentary diseases.


Assuntos
Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Melanoma Experimental/patologia , Glicoproteínas de Membrana/metabolismo , Oxirredutases/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Oxirredutases Intramoleculares/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Oxirredutases/genética , Fosforilação , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
4.
Protein Cell ; 11(10): 723-739, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32754890

RESUMO

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Oxirredutases/antagonistas & inibidores , Pandemias , Pneumonia Viral/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Infecções por Coronavirus/virologia , Crotonatos/farmacologia , Síndrome da Liberação de Citocina/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Humanos , Vírus da Influenza A/efeitos dos fármacos , Leflunomida/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/uso terapêutico , Oxirredutases/metabolismo , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Pirimidinas/biossíntese , Vírus de RNA/fisiologia , Relação Estrutura-Atividade , Toluidinas/farmacologia , Ubiquinona/metabolismo , Replicação Viral/efeitos dos fármacos
5.
Science ; 369(6499): 59-64, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32631887

RESUMO

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3' dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.


Assuntos
Cobre/metabolismo , Histonas/química , Oxirredutases/química , Multimerização Proteica , Animais , Biocatálise , Domínio Catalítico/genética , Mutação com Ganho de Função , Histonas/genética , Histonas/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química , Fatores de Transcrição/metabolismo , Xenopus laevis
6.
Nature ; 585(7824): 288-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32641834

RESUMO

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.


Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Ubiquinona/análogos & derivados , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ciona intestinalis/enzimologia , Ciclo do Ácido Cítrico , Citosol/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/deficiência , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Lactobacillus brevis/enzimologia , Masculino , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Neoplasias/enzimologia , Fosforilação Oxidativa , Oxirredutases/genética , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ubiquinona/metabolismo
7.
Food Chem ; 333: 127492, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659673

RESUMO

In this study, recombinant rice quiescin sulfhydryl oxidase (rQSOX) was expressed and characterized, and its performance in flour-processing quality was further evaluated. The purified rQSOX exhibited the highest sulfhydryl oxidation activity (1.96 IU/mg) using dithiothreitol as a substrate, accompanying the production of H2O2. The optimal temperature and pH were 60 °C and pH 8.0 for rQSOX catalyzing oxidation of dithiothreitol. And rQSOX retained 50% of its maximum activity after incubation at 80 °C for 1 h. Moreover, rQSOX supplementation improved the farinograph properties of dough, indicated by the increased dough stability time and decreased degree of softening, and enhanced viscoelastic properties of the dough. Addition of rQSOX (10 IU/g flour) provided remarkable improvement in specific volume (37%) and springiness (17%) of the steamed bread, and significantly reduced the hardness by half, which was attributed to the strengthened gluten network. The results provide an understanding for rQSOX using in flour-processing industry.


Assuntos
Farinha/análise , Oryza/enzimologia , Oxirredutases/química , Proteínas de Plantas/química , Triticum/química , Biocatálise , Pão/análise , Manipulação de Alimentos , Glutens/química , Dureza , Peróxido de Hidrogênio/análise , Oryza/química , Oryza/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
8.
J Am Mosq Control Assoc ; 36(1): 22-32, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32497474

RESUMO

In several insect species, resistance to pyrethroids and DDT (dichlorodiphenyltrichloroethane) is linked to point mutations in the voltage-gated sodium channel (VGSC) gene. Pyrethroid-based insecticides prolong the opening of sodium channels, causing paralysis known as a "knockdown" effect before mortality occurs. Point mutations in the VGSC gene result in decreased pyrethroid binding and reduced sensitivity to the insecticide-this resistance mechanism is known as knockdown resistance (kdr) as insects do not die but recover from paralysis with time. In Culex mosquito species loss of target site sensitivity to pyrethroids is linked to a number of substitutions, one of which is leucine (L) to phenylalanine (F) at residue 1014 (L1014F) in the VGSC gene. Here we report the identification of kdr-associated pyrethroid resistance and developing resistance in Cx. quinquefasciatus field collections from Collier County, FL. Evaluation of position 1014 of the VGSC in Cx. quinquefasciatus collections from 7 locations in Collier County, FL, revealed a wide range of genotypes from one part of the district to the other. Centers for Disease Control and Prevention bottle bioassay, linear regression analysis, and cage trial evaluations suggest that the L1014F mutation plays a role, at least in part, to the pyrethroid resistance status of Cx. quinquefasciatus collected in Collier County, FL. Furthermore, we identified resistance attributed to both oxidase and esterase activity, indicating that multiple mechanisms are responsible for pyrethroid resistance in Collier County Cx. quinquefasciatus.


Assuntos
Culex/genética , Esterases/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Oxirredutases/genética , Piretrinas/farmacologia , Animais , Culex/efeitos dos fármacos , Culex/enzimologia , Esterases/metabolismo , Feminino , Florida , Oxirredutases/metabolismo
9.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
10.
PLoS Genet ; 16(6): e1008838, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32544191

RESUMO

Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H202, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H202 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Peróxidos/metabolismo , Vulva/crescimento & desenvolvimento , Proteínas ras/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Mutação com Ganho de Função , Genes de Helmintos/genética , Oxirredução , Oxirredutases/metabolismo , Peróxidos/análise , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo
11.
PLoS One ; 15(6): e0235204, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584884

RESUMO

Manipulative strategies of ROS in cancer are often exhibited as changes in the redox and thiol ratio of the cells. Cellular responses to oxidative insults are generated in response to these changes which are triggered due to the rerouting of the metabolic framework to maintain survival under stress. However, mechanisms of these metabolic re-routing are not clearly understood and remained debatable. In the present work, we have designed a context-based dynamic metabolic model to establish that the coordinated functioning of glutathione peroxidase (GTHP), glutathione oxidoreductase (GTHO) and NADPH oxidase (NOX) is crucial in determining cancerous transformation, specifically in gliomas. Further, we propose that the puzzling duality of ROS (represented by changes in h2o2 in the present model) in exhibiting varying cellular fates can be determined by considering simultaneous changes in nadph/nadp+ and gsh/gssg that occur during the reprogramming of metabolic reactions. This will be helpful in determining the pro-apoptotic or anti-apoptotic fate of gliomas and can be useful in designing effective pro-oxidant and/or anti-oxidant therapeutic approaches against gliomas.


Assuntos
Apoptose , Glioma/metabolismo , Glutationa/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo , Glioma/patologia , Humanos , Proteínas de Neoplasias/metabolismo , Oxirredutases/metabolismo
12.
Pol J Microbiol ; 69(2): 205-215, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32548989

RESUMO

Oxidative stress-induced series of related degenerative diseases have received widespread attention. To screen new lactic acid bacteria (LAB) strains to resist oxidative stress, traditional Chinese fermented vegetables were used as a resource library to screen of LAB. The Lactobacillus fermentum JX306 strain, which showed high scavenging activity of DPPH free radical and hydrogen radical, and a strong lipid peroxidation inhibition rate in vitro was selected. L. fermentum JX306 was also examined for its antioxidant capacity in D-galactose-induced aging mice. The results showed that L. fermentum JX306 could significantly decrease malondialdehyde (MDA) levels and improve the activity of glutathione peroxidase (GSH-Px), and total antioxygenic capacity (TOC) in the serum, kidney, and liver. Meanwhile, the strain could remarkably upregulate the transcriptional level of the antioxidant-related enzyme genes, such as peroxiredoxin1 (Prdx1), glutathione reductase (Gsr), glutathione peroxidase (Gpx1), and thioredoxin reductase (TR3) encoding genes in the liver. Besides, histopathological observation proves that this probiotic strain could effectively inhibit oxidative damage to the liver and kidney in aging mice. Therefore, this unique antioxidant strain may have a high application value in the functional food industry and medicine industry.Oxidative stress-induced series of related degenerative diseases have received widespread attention. To screen new lactic acid bacteria (LAB) strains to resist oxidative stress, traditional Chinese fermented vegetables were used as a resource library to screen of LAB. The Lactobacillus fermentum JX306 strain, which showed high scavenging activity of DPPH free radical and hydrogen radical, and a strong lipid peroxidation inhibition rate in vitro was selected. L. fermentum JX306 was also examined for its antioxidant capacity in D-galactose-induced aging mice. The results showed that L. fermentum JX306 could significantly decrease malondialdehyde (MDA) levels and improve the activity of glutathione peroxidase (GSH-Px), and total antioxygenic capacity (TOC) in the serum, kidney, and liver. Meanwhile, the strain could remarkably upregulate the transcriptional level of the antioxidant-related enzyme genes, such as peroxiredoxin1 (Prdx1), glutathione reductase (Gsr), glutathione peroxidase (Gpx1), and thioredoxin reductase (TR3) encoding genes in the liver. Besides, histopathological observation proves that this probiotic strain could effectively inhibit oxidative damage to the liver and kidney in aging mice. Therefore, this unique antioxidant strain may have a high application value in the functional food industry and medicine industry.


Assuntos
Microbiologia de Alimentos , Lactobacillus fermentum/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Envelhecimento , Animais , Alimentos e Bebidas Fermentados/microbiologia , Galactose/farmacologia , Camundongos , Estresse Oxidativo/efeitos dos fármacos
13.
Nature ; 584(7819): 69-74, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32512577

RESUMO

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.


Assuntos
Alcenos/química , Alcenos/metabolismo , Hidrogênio/química , Hidrogênio/metabolismo , Luz , Oxirredutases/metabolismo , Processos Fotoquímicos/efeitos da radiação , Álcoois/química , Álcoois/metabolismo , Alquilação/efeitos da radiação , Biocatálise/efeitos da radiação , Biomassa , Carboxiliases/metabolismo , Flavinas/metabolismo , Modelos Químicos , Modelos Moleculares , Estereoisomerismo
14.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(3): 290-293, 2020 Apr 26.
Artigo em Chinês | MEDLINE | ID: mdl-32468792

RESUMO

OBJECTIVE: To evaluate the effects of Cu2+ and Cd2+ at different concentrations on superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activity in Oncomelania hupensis. METHODS: Cu2+- and Cd2+-containing solutions were prepared at 7 concentrations, and O. hupensis snails were exposed to the solutions for 24 h, of 15 snails in each concentration. Then, the snail body was collected following removal of the snail shell and homogenated, and the SOD, CAT and POS activities were detected in the supernatants. RESULTS: With the increase of the Cu2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline-rise-decline tendency, while the POD activity showed a tendency towards rise followed by decline. With the increase of the Cd2+ concentration, the SOD activity appeared a rise followed by a reduction in O. hupensis snails, and the CTA activity appeared a decline- rise- decline tendency, while the POD activity showed a decline-rise-decline tendency. CONCLUSIONS: Exposure to Cu2+ and Cd2+ at high concentrations results in a decline in the activity of SOD, CAT and POD in O. hupensis at the same time.


Assuntos
Cádmio , Cobre , Oxirredutases , Caramujos , Animais , Cádmio/toxicidade , Catalase/metabolismo , Cobre/toxicidade , Ativação Enzimática/efeitos dos fármacos , Íons/toxicidade , Oxirredutases/metabolismo , Peroxidase/metabolismo , Caramujos/efeitos dos fármacos , Caramujos/enzimologia , Superóxido Dismutase/metabolismo
15.
Chemosphere ; 253: 126739, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464773

RESUMO

Biological denitrification is a promising and green technology for air pollution control. To investigate the nitric oxide reductase (NOR) that dominates NO reduction efficiency in biological purification, the heterologous prokaryotic expression system of the norB gene, which encodes the core peptide of the catalytic reduction structure in the NOR from Achromobacter denitrificans strain TB, was constructed in Escherichia coli BL21 (DE3). Results showed that the 1218 bp-long norB gene was expressed at the highest level under 1.0 mM IPTG for 5 h at 30 °C, and the relative expression abundance of norB in recombinant E. coli was increased by 16.6 times compared with that of the wild-type TB. However, the NO reduction efficiency and NOR activity of strain TB was 2.7 and 1.83 times higher than those of recombinant E. coli, respectively. On the basis of genomic reassembly and protein structure modeling, the core peptide of the NOR catalytic reduction structure from Achromobacter sp. TB can independently exert NO reduction. The low NO degradation efficiency of recombinant E. coli may be due to the lack of a NorC-like structure that increases the enzyme activity of the NorB protein. The results of this study can be used as basis for further research on the structure and function of NOR.


Assuntos
Achromobacter denitrificans/metabolismo , Oxirredutases/metabolismo , Catálise , Desnitrificação , Escherichia coli/metabolismo , Óxido Nítrico/metabolismo , Peptídeos
16.
Chemosphere ; 253: 126654, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32464761

RESUMO

Heme oxygenase-1(HO-1) is a stress-inducible enzyme that mediates antioxidative and cytoprotective effects to maintain cellular redox homeostasis. In the present study, the full sequence of HO-1 was cloned from golden pompano(Trachinotus ovatus) by RT-PCR and RACE-PCR. The full cDNA sequence of HO-1 was 1349 bp in length which comprised of a 726 bp open reading frame (ORF) preceded by 262 bp 5'-untranslated region (UTR), and followed by a 360 bp 3'UTR, encoding 241 amino acid residues. Phylogenetic analysis revealed that HO-1 showed highest similarity to that of Takifugu rubripes. Tissue distribution analysis showed that the expression level of HO-1 was relatively high in heart, liver and spleen. A trial was conducted to investigate the response of Nrf2/HO-1 signaling pathway to oxidative stress induced by copper. The results showed that mRNA expression of NF-E2-related nuclear factor2 (Nrf2), Kelch-like-ECH-associated protein1 (keap1), superoxide dismutase (SOD), catalase (CAT), HO-1, NAD(P)H quinone oxidoreductase 1 (NQO1) and Glutathione peroxidase (GSH-PX) all significantly increased in copper treated group than that in the control group. This work provides new insight into the molecular mechanism underlying the Nrf2/HO-1 pathway in oxidative response in T. ovatus.


Assuntos
Cobre/farmacologia , Heme Oxigenase-1/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Perciformes/metabolismo , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Heme Oxigenase-1/metabolismo , Oxirredutases/metabolismo , Análise de Sequência de DNA , Distribuição Tecidual
17.
Proc Natl Acad Sci U S A ; 117(17): 9349-9355, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291342

RESUMO

Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Oxirredutases/química , Bactérias/metabolismo , Cobre/química , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Transporte de Íons , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxigênio/metabolismo , Bombas de Próton/metabolismo , Prótons , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Chemistry ; 26(22): 4884, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32297426

RESUMO

Invited for the cover of this issue is the group of Ulrich Schwaneberg at the Institute of Biotechnology, RWTH-Aachen University and DWI Lebniz Institute of Interactive Materials. The picture calls for special attention to be paid to the extra Cu binding site of Copper efflux Oxidase (CueO), due to its predominant function in tuning the electrocatalytic kinetics towards oxygen reduction. Read the full text of the article at 10.1002/chem.201905598.


Assuntos
Ceruloplasmina/metabolismo , Lacase/química , Oxirredutases/química , Sítios de Ligação , Ceruloplasmina/química , Transporte de Elétrons , Proteínas de Escherichia coli/química , Cinética , Oxirredução , Oxirredutases/metabolismo
19.
PLoS One ; 15(4): e0231965, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324772

RESUMO

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen's aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism.


Assuntos
Materiais Biomiméticos , Meios de Cultura , Oxirredutases/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Urina , Aerobiose , Transporte de Elétrons , NADH Desidrogenase/metabolismo , Quinonas/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-32234661

RESUMO

Cadmium (Cd) and zinc (Zn) coexist in the environment but interact differently in plants. Cosmos bipinnatus has been potentially considered as a Cd-accumulator. Thus, this study investigated the detoxification mechanism in C. bipinnatus seedlings under Cd, Zn and Cd + Zn stresses. In the present study, the presence of Zn inhibited Cd uptake and translocation, whereas Cd merely hindered Zn uptake. The concentration of Cd in soluble fraction significantly decreased and Cd was bounded to the cell wall in root under Cd + Zn stress. Meanwhile, Zn and Cd mutually decreased their concentrations in the ethanol extractable form (FE) and water extractable form (FW) in roots and shoots. Furthermore, Cd + Zn stress enhanced the activities of superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) compared to Cd stress alone. These results suggested that Zn effectively decreased Cd uptake and translocation, changed their subcellular distributions, regulated their chemical forms composition and increased antioxidative enzyme activities, thereby enhancing the tolerance to Cd in C. bipinnatus. This study physiologically revealed the interactive effect of Cd and Zn on the detoxification mechanism of Cd in C. bipinnatus and provided new information on phytoremediation of the heavy metal contaminated soils.


Assuntos
Asteraceae/efeitos dos fármacos , Cádmio , Plântula , Poluentes do Solo , Estresse Fisiológico , Zinco , Asteraceae/metabolismo , Cádmio/metabolismo , Oxirredutases/metabolismo , Raízes de Plantas/efeitos dos fármacos , Plântula/efeitos dos fármacos , Poluentes do Solo/toxicidade , Estresse Fisiológico/efeitos dos fármacos , Zinco/toxicidade
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