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1.
Mar Drugs ; 17(5)2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31058830

RESUMO

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography-mass spectrometry (LC-MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC-MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.


Assuntos
Bacillus/genética , Bactérias/genética , Gastrópodes/microbiologia , Peroxidases/genética , Animais , Bacillus/isolamento & purificação , Bacillus/metabolismo , Bactérias/isolamento & purificação , Bactérias/metabolismo , Região Branquial/metabolismo , Região Branquial/microbiologia , Indóis/análise , Moluscos , Análise de Sequência de RNA , Simbiose
2.
J Agric Food Chem ; 67(19): 5486-5495, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31012315

RESUMO

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.


Assuntos
Cannabis/microbiologia , Proteínas Fúngicas/metabolismo , Pleurotus/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Glicosilação , Lacase/química , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Caules de Planta/microbiologia , Pleurotus/classificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Transporte Proteico
3.
Int J Mol Sci ; 20(6)2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875904

RESUMO

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.


Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Leishmania braziliensis/enzimologia , Serina Proteases/genética , Serina Proteases/metabolismo , Cromatografia de Afinidade , Simulação por Computador , Regulação da Expressão Gênica , Leishmania braziliensis/genética , Peso Molecular , Peroxidases/genética , Peroxidases/metabolismo , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sensibilidade e Especificidade , Subtilisina/genética , Subtilisina/metabolismo
4.
Gene ; 700: 149-162, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30904716

RESUMO

Class III peroxidases are classical secretory plant peroxidases belonging to a large multi-gene family. Class III peroxidases are involved in various physical processes and the response to biotic and abiotic stress to protect plants from environmental adversities. In this study, 151 BdPrx genes were identified using HMM and Blastp program. According to their physical location, the 151 BdPrx genes were mapped on five chromosomes. The results of Gene Structure Display Serve and MEME revealed that BdPrxs in the same subgroup shared similar gene structure, and their protein sequences were highly conserved. Based on the analysis of evolutionary relationships and Ka/Ks, 151 BdPrx genes were divided into 15 subgroups, they have undergone purifying selection. In addition, the result of GO annotation showed that 100% of the BdPrxs participated in antioxidant. The protein-protein interaction network was constructed using the orthology-based method, found that 66 BdPrxs were involved in the regulatory network and 183 network branches were identified. Furthermore, analysis of the transcriptome data indicated that the BdPrx genes responded to low concentration of exogenous phytohormones and exhibited different levels of expression in the different tissues. Subsequently, 19 genes were selected for quantitative real-time PCR and found to be mainly expressed in the roots, might preferentially respond to hydrogen peroxide and gibberellin. Our results provide a foundation for further evolutionary and functional study of Prx genes in B. distachyon.


Assuntos
Brachypodium/enzimologia , Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Peroxidases/genética , Brachypodium/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Anotação de Sequência Molecular , Família Multigênica , Peroxidases/metabolismo , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Distribuição Tecidual
5.
Nat Commun ; 10(1): 1101, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846684

RESUMO

Bacterial diheme peroxidases represent a diverse enzyme family with functions that range from hydrogen peroxide (H2O2) reduction to post-translational modifications. By implementing a sequence similarity network (SSN) of the bCCP_MauG superfamily, we present the discovery of a unique diheme peroxidase BthA conserved in all Burkholderia. Using a combination of magnetic resonance, near-IR and Mössbauer spectroscopies and electrochemical methods, we report that BthA is capable of generating a bis-Fe(IV) species previously thought to be a unique feature of the diheme enzyme MauG. However, BthA is not MauG-like in that it catalytically converts H2O2 to water, and a 1.54-Å resolution crystal structure reveals striking differences between BthA and other superfamily members, including the essential residues for both bis-Fe(IV) formation and H2O2 turnover. Taken together, we find that BthA represents a previously undiscovered class of diheme enzymes, one that stabilizes a bis-Fe(IV) state and catalyzes H2O2 turnover in a mechanistically distinct manner.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/enzimologia , Hemeproteínas/metabolismo , Peroxidases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Burkholderia/genética , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Hemeproteínas/química , Hemeproteínas/genética , Ferro/química , Cinética , Modelos Moleculares , Oxirredução , Peroxidases/química , Peroxidases/genética , Conformação Proteica
6.
World J Microbiol Biotechnol ; 35(4): 53, 2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30900038

RESUMO

The oxidative stress response of the highly resistant actinomycete Dietzia cinnamea P4 after treatment with hydrogen peroxide (H2O2) was assessed in order to depict the possible mechanisms underlying its intrinsic high resistance to DNA damaging agents. We used transcriptional profiling to monitor the magnitude and kinetics of changes in the mRNA levels after exposure to different concentrations of H2O2 at 10 min and 1 h following the addition of the stressor. Catalase and superoxide dismutase genes were induced in different ways, according to the condition applied. Moreover, alkyl hydroperoxide reductase ahpCF, thiol peroxidase, thioredoxin and glutathione genes were upregulated in the presence of H2O2. Expression of peroxidase genes was not detected during the experiment. Overall results point to an actinomycete strain endowed with a set of enzymatic defenses against oxidative stress and with the main genes belonging to a functional SOS system (lexA, recA, uvrD), including suppression of lexA repressor, concomitantly to recA and uvrD gene upregulation upon H2O2 challenge.


Assuntos
Actinomycetales/efeitos dos fármacos , Actinomycetales/metabolismo , Peróxido de Hidrogênio/efeitos adversos , Estresse Oxidativo , Resposta SOS (Genética)/fisiologia , Actinomycetales/enzimologia , Actinomycetales/genética , Proteínas de Bactérias/genética , Catalase/classificação , Catalase/genética , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Glutationa/genética , Cinética , Peroxidases/genética , Peroxirredoxinas/genética , Filogenia , RNA Mensageiro/metabolismo , Recombinases Rec A/genética , Resposta SOS (Genética)/genética , Análise de Sequência , Serina Endopeptidases/genética , Superóxido Dismutase/genética , Tiorredoxinas/genética , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
7.
Enzyme Microb Technol ; 123: 21-29, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30686347

RESUMO

Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7-8 fold enhanced kcat/KM towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin. Residues near the predicted Mn(II) binding site were also investigated by site-directed mutagenesis, and mutants S223 N and H127R showed 4-7-fold increased kcat/KM for Mn(II) oxidation. Mutant F128R also showed enhanced thermostability, compared to wild-type Dyp1B. Testing of mutants for low molecular weight product release from Protobind alkali lignin revealed that mutant H169 L showed enhanced product release, compared with WT enzyme, and the formation of three low molecular weight metabolites by this mutant was detected by reverse phase HPLC analysis.


Assuntos
Lignina/química , Peroxidases/química , Peroxidases/metabolismo , Fenóis/química , Engenharia de Proteínas , Pseudomonas fluorescens/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Corantes/química , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Peroxidases/genética , Conformação Proteica , Homologia de Sequência
8.
Arch Microbiol ; 200(8): 1267-1278, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29947838

RESUMO

Overcoming recalcitrance of lignin has motivated bioprospecting of high-yielding enzymes from environmental ligninolytic microorganisms associated with lignocellulose degrading-systems. Here, we performed isolation of 21 ligninolytic strains belonging to the genus Klebsiella spp., driven by the presence of lignin in the media. The fastest-growing strains (FP10-5.23, FP10-5.22 and P3TM1) reached the stationary phase in approximately 24 h, in the media containing lignin as the main carbon source. The strains showed biochemical evidence of ligninolytic potential in liquid- and solid media-converting dyes, which the molecular structures are similar to lignin fragments. In liquid medium, higher levels of dye decolorization was observed for P3TM.1 in the presence of methylene blue, reaching 98% decolorization in 48 h. The highest index values (1.25) were found for isolates P3TM.1 and FP10-5.23, in the presence of toluidine blue. The genomic analysis revealed the presence of more than 20 genes associated with known prokaryotic lignin-degrading systems. Identification of peroxidases (lignin peroxidase-LiP, dye-decolorizing peroxidase-DyP, manganese peroxidase-MnP) and auxiliary activities (AA2, AA3, AA6 and AA10 families) among the genetic repertoire suggest the ability to produce extracellular enzymes able to attack phenolic and non-phenolic lignin structures. Our results suggest that the Klebsiella spp. associated with fresh water and soil may play important role in the cycling of recalcitrant molecules in the Caatinga (desert-like Brazilian biome), and represent a potential source of lignin-degrading enzymes with biotechnological applications.


Assuntos
Klebsiella/enzimologia , Klebsiella/metabolismo , Lignina/metabolismo , Peroxidases/genética , Biodegradação Ambiental , Brasil , Água Doce/microbiologia , Klebsiella/classificação , Klebsiella/genética , Oxirredutases/genética , Microbiologia do Solo
9.
Biomed Res Int ; 2018: 3828627, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29850510

RESUMO

The recombinant catalase-peroxidase HPI from E. coli was used as an alternative enzyme in polymerization reactions for the production of (-) epicatechin oligomers and their biological activity was characterized. The enzyme was prepared in two forms: a purified and an immobilized form. Both were tested for their activity in oxidative polymerization reactions, and their stability and reusability were assessed. The polymerization reactions were followed by SEC-HPLC analyses, and the substrate was completely converted into one or more polymerization products depending on the reactions conditions. Results showed that the utilized conditions allowed for the isolation of some oligomers of different molecular weight: the oligomers containing 6 and 7 units of epicatechin substrate are the heaviest ones. Epicatechin was also used in reactions catalyzed by HRP in the same reaction conditions for comparison. In addition, one selected oligomer obtained by HPI enzyme catalysis was shown to act as in vitro inhibitor of tumor cell growth, like one oligomer deriving from epicatechin by HRP catalysis. These data confirm that epicatechin oligomeric form is more effective than its monomer in biological activity and suggest the use of HPI as an alternative enzyme in reactions for the production of epicatechin oligomers.


Assuntos
Proteínas de Bactérias/metabolismo , Peroxidases/metabolismo , Polifenóis/metabolismo , Polifenóis/farmacologia , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catequina/química , Catequina/metabolismo , Catequina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Humanos , Oxirredução , Peroxidases/química , Peroxidases/genética , Polifenóis/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
10.
Ann Diagn Pathol ; 34: 161-165, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29661721

RESUMO

BACKGROUND: Peroxidasin (PXDN) is an extracellular matrix protein with peroxidase activity. PXDN has been reported to participate in the processes of epithelial mesenchymal transition. However, the roles of PXDN in progression of cancers are still rare. METHODS: Expression profiles of PXDN in ovarian cancer (OC) tissues were obtained from GEO and TCGA database. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression of PXDN in OC cells. Kaplan-Meier method was used to analyze the overall survival of OC patients. Furthermore, effects of PXDN knockdown on the proliferation, invasion as well as migration of HEY cells were examined by Cell Counting kit-8 (CCK-8), wound healing and transwell assay. Additionally, western blot assay was conducted to detect the levels of several key proteins in PI3K/Akt pathway. RESULTS: PXDN was highly expressed in OC tissues and cells. OC Patients with high PXDN expression showed poorer overall survival rate compared to the OC patients with low PXDN expression. The results of the present study demonstrated that knockdown of PXDN significantly suppressed the proliferation, invasion and migration of HEY cells. In addition, after silencing PXDN in HEY cells, the expression levels of the key protein phosphorylation in PI3K/Akt pathway were obviously decreased, including p-PI3K and p-Akt, that resulting in the inhibition of PI3K/Akt pathway activation. CONCLUSION: PXDN might play a promoter role in the proliferation, invasion and migration of OC cells through regulating the activation of PI3K/Akt pathway. Therefore, PXDN might be regarded as a potential target for OC therapy.


Assuntos
Neoplasias Ovarianas/diagnóstico , Peroxidases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Peroxidases/genética , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética
11.
DNA Cell Biol ; 37(6): 524-534, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29596001

RESUMO

Although date palm is a relatively salt-tolerant plant, the molecular basis of this tolerance is complex and poorly understood. Therefore, this study aimed to identify the genes involved in salinity tolerance using a basic yeast functional bioassay. To achieve this, a date palm cDNA library was overexpressed in Saccharomyces cerevisiae cells. The expression levels of selected genes that make yeast cells tolerant to salt were subsequently validated in the leaf and root tissues of date palm seedlings using a quantitative PCR method. About 6000 yeast transformant cells were replica printed and screened on a synthetic minimal medium containing 1.0 M of NaCl. The screening results showed the presence of 62 salt-tolerant transformant colonies. Sequence analysis of the recombinant yeast plasmids revealed the presence of a group of genes with potential salt-tolerance functions, such as aquaporins (PIP), serine/threonine protein kinases (STKs), ethylene-responsive transcription factor 1 (ERF1), and peroxidases (PRX). The expression pattern of the selected genes endorsed the hypothesis that these genes may be involved in salinity tolerance, as they showed a significant (p < 0.05) overexpression trend in both the leaf and root tissues in response to salinity. The genes identified in this project are suitable candidates for the further functional characterization of date palms.


Assuntos
Biblioteca Gênica , Genes de Plantas/genética , Phoeniceae/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal/genética , Aquaporinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peroxidases/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Plantas Tolerantes a Sal/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética
12.
Biochim Biophys Acta Bioenerg ; 1859(6): 411-422, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29550214

RESUMO

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be -170 mV, +133 mV and +210 mV, respectively, at pH 7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH 7.0 and millimolar range KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM = 0.6 ±â€¯0.2 and 1.8 ±â€¯0.5 mM H2O2, respectively), with similar turnover numbers (kcat = 19 ±â€¯2 and 13 ±â€¯2 s-1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV) = O species was observed upon incubation of fully oxidized YhjA with H2O2. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.


Assuntos
Citocromo-c Peroxidase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Heme/química , Peróxido de Hidrogênio/química , Hidroquinonas/química , Peroxidases/química , Sítios de Ligação , Biocatálise , Clonagem Molecular , Citocromo-c Peroxidase/genética , Citocromo-c Peroxidase/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Hidroquinonas/metabolismo , Cinética , Oxirredução , Peroxidases/genética , Peroxidases/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
Chin J Nat Med ; 16(3): 203-209, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29576056

RESUMO

Caesalpinia sappan L., belonging to the family Leguminosae, is a medicinal plant that is distributed in Southeast Asia. The dried heartwood of this plant is used as a traditional ingredient of food, red dyes, and folk medicines in the treatment of diarrhea, dysentery, tuberculosis, skin infections, and inflammation. Brazilin is the major active compound, which has exhibited various pharmacological effects, including anti-platelet activity, anti-hepatotoxicity, induction of immunological tolerance, and anti-inflammatory and antioxidant activities. The present study aimed to evaluate the antioxidant activity and expression of antioxidant enzymes of C. sappan L. extract and its major compound, brazilin, in human epidermal keratinocytes exposed to UVA irradiation. Our results indicated that C. sappan L. extract reduced UVA-induced H2O2 production via GPX7 activation. Moreover, brazilin exhibited antioxidant effects that were similar to those of C. sappan L. via glutathione peroxidase 7 (GPX7), suggesting that C. sappan L. extract and its natural compound represent potential treatments for oxidative stress-induced photoaging of skin.


Assuntos
Benzopiranos/farmacologia , Caesalpinia/química , Queratinócitos/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidases/genética , Extratos Vegetais/farmacologia , Substâncias Protetoras/farmacologia , Antioxidantes/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Peroxidases/metabolismo , Raios Ultravioleta
14.
J Inorg Biochem ; 181: 65-73, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29407909

RESUMO

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S2 = 0.87 ±â€¯0.03 and S2 = 0.76 ±â€¯0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ±â€¯1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.


Assuntos
Hemoglobinas/metabolismo , Modelos Moleculares , Peroxidases/metabolismo , Poliquetos/enzimologia , Algoritmos , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Hemoglobinas/química , Hemoglobinas/genética , Histidina/química , Histidina/genética , Histidina/metabolismo , Cinética , Ligantes , Simulação de Dinâmica Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peroxidases/química , Peroxidases/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Xenônio/química , Xenônio/metabolismo
15.
BMC Evol Biol ; 18(1): 18, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29422028

RESUMO

BACKGROUND: Peroxiredoxins are ubiquitous thiol-dependent peroxidases that represent a major antioxidant defense in both prokaryotic cells and eukaryotic organisms. Among the six vertebrate peroxiredoxin isoforms, peroxiredoxin-5 (PRDX5) appears to be a particular peroxiredoxin, displaying a different catalytic mechanism, as well as a wider substrate specificity and subcellular distribution. In addition, several evolutionary peculiarities, such as loss of subcellular targeting in certain species, have been reported for this enzyme. RESULTS: Western blotting analyses of 2-cys PRDXs (PRDX1-5) failed to identify the PRDX5 isoform in chicken tissue homogenates. Thereafter, via in silico analysis of PRDX5 orthologs, we went on to show that the PRDX5 gene is conserved in all branches of the amniotes clade, with the exception of aves. Further investigation of bird genomic sequences and expressed tag sequences confirmed the disappearance of the gene, though TRMT112, a gene located closely to the 5' extremity of the PRDX5 gene, is conserved. Finally, using in ovo electroporation to overexpress the long and short forms of human PRDX5, we showed that, though the gene is lost in birds, subcellular targeting of human PRDX5 is conserved in the chick. CONCLUSIONS: Further adding to the distinctiveness of this enzyme, this study reports converging evidence supporting loss of PRDX5 in aves. In-depth analysis revealed that this absence is proper to birds as PRDX5 appears to be conserved in non-avian amniotes. Finally, taking advantage of the in ovo electroporation technique, we validate the subcellular targeting of human PRDX5 in the chick embryo and bring forward this gain-of-function model as a potent way to study PRDX5 functions in vivo.


Assuntos
Galinhas/metabolismo , Peroxirredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular Tumoral , Sequência Conservada , Cisteína/metabolismo , Eletroporação , Humanos , Peroxidases/genética , Peroxirredoxinas/química , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo
16.
Microbiol Immunol ; 62(3): 158-167, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29345334

RESUMO

Three different katG sequences (katGI, katGII and katGIII) were identified in the Mycobacterium smegmatis genome. The contributions of the three katG genes to survival of the bacterium were examined by constructing disruptants of these three genes. The katGIII sequence did not produce a functional catalase-peroxidase. Analyses of peroxidase activity and mRNA expression revealed that in wild type M. smegmatis, expression dominance between KatGI and KatGII was switched in the exponential and stationary growth phases. Susceptibility of the M. smegmatis gene disruptants to hydrogen peroxide (H2 O2 ) was tested in two growth phases. In the exponential phase, the katGI-null strain was more susceptible to H2 O2 than the katGII-null strain, indicating that KatGI plays a more important role in survival than KatGII in this growth phase. In contrast, in the stationary phase, growth of the katGII-null strain was inhibited at lower concentrations of H2 O2 . These results suggest that M. smegmatis has two types of catalase-peroxidases, expressions of which are controlled under different gene regulatory systems. Isoniazid (INH) susceptibilities of the katG-null strains were also examined and it was found that katGI is a major determinant of M. smegmatis susceptibility to INH.


Assuntos
Proteínas de Bactérias/genética , Catalase/genética , Catalase/fisiologia , Genes Bacterianos/genética , Isoniazida/farmacologia , Mycobacterium smegmatis/genética , Peroxidases/genética , Peróxidos/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Peróxido de Hidrogênio/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/crescimento & desenvolvimento , Estresse Oxidativo , RNA Mensageiro , Análise de Sequência de DNA , Transcrição Genética/efeitos dos fármacos
17.
Gene ; 646: 195-202, 2018 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-29305973

RESUMO

Peroxidasin (PXDN), an ECM protein with peroxidase activity, is integral to basement membrane consolidation through catalysis of sulfilimine bonds in collagen IV. PXDN is also involved in processes where epithelial-mesenchymal transition (EMT) takes place, namely fibrosis, development and cancer. We therefore investigated whether PXDN is regulated by the EMT-master-regulator, Snai1. During TGF-ß1-induced EMT, PXDN expression decreased by up to 47% in two cervical-carcinoma cell lines, with concomitant increases in Snai1 and vimentin, and decrease in E-cadherin. TGF-ß1 induced Snai1 binding to the PXDN promoter (as assessed by chromatin immunoprecipitation-PCR) and significantly repressed luciferase reporter gene expression, as did Snai1 overexpression. In summary, our findings show that Snai1 mediates repression of PXDN and consolidate a role for this ECM-modifier during EMT.


Assuntos
Transição Epitelial-Mesenquimal , Peroxidases/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Peroxidases/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
18.
Redox Biol ; 15: 297-315, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29304480

RESUMO

The system (PTTRS) formed by typical 2-Cys peroxiredoxins (Prx), thioredoxin (Trx), Trx reductase (TrxR), and sulfiredoxin (Srx) is central in antioxidant protection and redox signaling in the cytoplasm of eukaryotic cells. Understanding how the PTTRS integrates these functions requires tracing phenotypes to molecular properties, which is non-trivial. Here we analyze this problem based on a model that captures the PTTRS' conserved features. We have mapped the conditions that generate each distinct response to H2O2 supply rates (vsup), and estimated the parameters for thirteen human cell types and for Saccharomyces cerevisiae. The resulting composition-to-phenotype map yielded the following experimentally testable predictions. The PTTRS permits many distinct responses including ultra-sensitivity and hysteresis. However, nearly all tumor cell lines showed a similar response characterized by limited Trx-S- depletion and a substantial but self-limited gradual accumulation of hyperoxidized Prx at high vsup. This similarity ensues from strong correlations between the TrxR, Srx and Prx activities over cell lines, which contribute to maintain the Prx-SS reduction capacity in slight excess over the maximal steady state Prx-SS production. In turn, in erythrocytes, hepatocytes and HepG2 cells high vsup depletes Trx-S- and oxidizes Prx mainly to Prx-SS. In all nucleated human cells the Prx-SS reduction capacity defined a threshold separating two different regimes. At sub-threshold vsup the cytoplasmic H2O2 concentration is determined by Prx, nM-range and spatially localized, whereas at supra-threshold vsup it is determined by much less active alternative sinks and µM-range throughout the cytoplasm. The yeast shows a distinct response where the Prx Tsa1 accumulates in sulfenate form at high vsup. This is mainly due to an exceptional stability of Tsa1's sulfenate. The implications of these findings for thiol redox regulation and cell physiology are discussed. All estimates were thoroughly documented and provided, together with analytical approximations for system properties, as a resource for quantitative redox biology.


Assuntos
Antioxidantes/metabolismo , Oxirredução , Peroxirredoxinas/metabolismo , Tiorredoxinas/metabolismo , Linhagem Celular Tumoral , Glutationa/genética , Glutationa/metabolismo , Células Hep G2 , Humanos , Estresse Oxidativo/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiorredoxina Redutase 1/metabolismo
19.
Arch Microbiol ; 200(1): 159-169, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28887685

RESUMO

A zinc (Zn)-tolerant fungus, designated BC109-2, was isolated from rhizosphere soil and was identified as Penicillium janthinellum BC109-2 based on ITS sequence analysis. To understand its Zn tolerance mechanisms, a series of studies was carried out addressing the subcellular distribution of Zn, its chemical forms, and the antioxidant system (superoxide dismutase, catalase, peroxidase, glutathione reductase, glutathione S-transferase, reduced glutathione, oxidized glutathione and malondialdehyde) of the fungus. The maximum level of resistance to Zn for strain BC109-2 is 2100 mg L-1. The Zn contents and percentages of cell wall and soluble fraction increased with increasing Zn concentration in the medium, which indicated extracellular accumulation/precipitation and vacuolar compartmentation mechanism might play significant role in the detoxificating process. The proportion of inactive forms of Zn was higher in the fungus, which indicated that BC109-2 mainly formed inactive Zn and stored it in the cell walls and vacuoles to decrease Zn toxicity. Furthermore, changes in antioxidant enzyme activities at various concentrations of Zn showed that the addition of Zn could cause oxidative stress in the fungal cells and that antioxidant enzymes in fungi played important roles in resistance to Zn toxicity. Moreover, the high level of lipid peroxidation showed that the protective effects of the antioxidant system were not sufficient at the high concentrations of Zn even though the antioxidant enzyme activity levels were very high. The purpose of this work is to figure out the heavy metal tolerance mechanisms of microorganisms in soil and the microbial isolate could be potentially used in bioremediation of Zn-contaminated environments.


Assuntos
Penicillium/isolamento & purificação , Penicillium/metabolismo , Zinco/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catalase/genética , Catalase/metabolismo , Glutationa/genética , Glutationa/metabolismo , Estresse Oxidativo , Penicillium/classificação , Penicillium/genética , Peroxidases/genética , Peroxidases/metabolismo , Solo/química , Microbiologia do Solo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
20.
Int J Biol Macromol ; 106: 636-646, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28813685

RESUMO

Two extracellular peroxidases from Bjerkandera adusta strain CX-9, namely a lignin peroxidase (called LiP BA45) and manganese peroxidase (called MnP BA30), were purified simultaneously by applying successively, ammonium sulfate precipitation-dialysis, Mono-S Sepharose anion-exchange and Sephacryl S-200 gel filtration and biochemically characterized. The sequence of their NH2-terminal amino acid residues showed high homology with those of fungi peroxidases. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzymes MnP BA30 and LiP BA45 were a monomers with a molecular masses 30125.16 and 45221.10Da, respectively. While MnP BA30 was optimally active at pH 3 and 70°C, LiP BA45 showed optimum activity at pH 4 and 50°C. The two enzymes were inhibited by sodium azide and potassium cyanide, suggesting the presence of heme-components in their tertiary structures. The Km and Vmax for LiP BA45 toward 2,4-Dichlorolphenol (2,4-DCP) were 0.099mM and 9.12U/mg, respectively and for MnP BA30 toward 2,6-Dimethylphenol (2,6-DMP), they were 0.151mM and 18.60U/mg, respectively. Interestingly, MnP BA30 and LiP BA45 demonstrated higher catalytic efficiency than that of other tested peroxidases (MnP, LiP, HaP4, and LiP-SN) and marked organic solvent-stability and dye-decolorization efficiency. Data suggest that these peroxidases may be considered as potential candidates for future applications in distaining synthetic-dyes.


Assuntos
Clorofenóis/metabolismo , Coriolaceae/enzimologia , Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Peroxidases/metabolismo , Xilenos/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Corantes/metabolismo , Coriolaceae/genética , Ensaios Enzimáticos , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Peroxidases/genética , Peroxidases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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