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1.
PLoS One ; 16(8): e0256376, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34437564

RESUMO

The use of potent fungal mixed cultures is a promising technique for the biodegradation of crude oil. Four isolates of fungi, namely, Alternaria alternata (AA-1), Aspergillus flavus (AF-3), Aspergillus terreus (AT-7), and Trichoderma harzianum (TH-5), were isolated from date palm soil in Saudi Arabia. The mixed fungal of the four isolates have a powerful tool for biodegradation up to 73.6% of crude oil (1%, w/v) in 14 days. The fungal consortium no. 15 containing the four isolates (1:1:1:1) performed significantly better as a biodegradation agent than other consortium in a variety of environmental factors containing crude oil concentration, incubation temperature, initial pH, biodegradation time and the salinity of the medium. The fungal consortium showed better performance in the biodegradation of normal alkanes (n-alkanes) than that of the polycyclic aromatic hydrocarbons (PAHs); the biodegradation efficiency of normal alkanes of the fungal consortium (67.1%) was clearly high than that of the PAHs (56.8%).


Assuntos
Fungos/fisiologia , Petróleo/microbiologia , 2,6-Dicloroindofenol/metabolismo , Análise de Variância , Biodegradação Ambiental , Fungos/enzimologia , Fungos/genética , Fungos/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Consórcios Microbianos , Petróleo/análise , Hidrocarbonetos Policíclicos Aromáticos/análise , Probabilidade , RNA Ribossômico 5,8S/genética , Rizosfera , Salinidade , Temperatura , Fatores de Tempo
2.
Biochemistry (Mosc) ; 85(8): 908-919, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33045951

RESUMO

Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M-1·s-1, (9 ± 2) × 104 M-1·s-1, and (1.6 ± 0.5) × 105 M-1·s-1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.


Assuntos
Di-Hidrolipoamida Desidrogenase/metabolismo , Elétrons , Miocárdio/metabolismo , NADH Desidrogenase/metabolismo , Zinco/metabolismo , 2,6-Dicloroindofenol/metabolismo , Animais , Domínio Catalítico , Escherichia coli/enzimologia , Peróxido de Hidrogênio/metabolismo , Cinética , Oxirredução , Superóxidos/metabolismo , Suínos , Tiorredoxina Dissulfeto Redutase/metabolismo
3.
J Biol Inorg Chem ; 25(2): 199-212, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32060650

RESUMO

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.


Assuntos
2,6-Dicloroindofenol/química , Oxirredutases do Álcool/química , Elétrons , Metilfenazônio Metossulfato/química , Fenazinas/química , Tetrametilfenilenodiamina/química , 2,6-Dicloroindofenol/metabolismo , Oxirredutases do Álcool/metabolismo , Methylobacterium extorquens/enzimologia , Metilfenazônio Metossulfato/metabolismo , Estrutura Molecular , Fenazinas/metabolismo , Tetrametilfenilenodiamina/metabolismo , Verrucomicrobia/enzimologia
4.
Photosynth Res ; 137(3): 421-429, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29767343

RESUMO

The peculiarities of interaction of cyanobacterial photosystem I with redox mediators 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were investigated. The higher donor efficiency of the reduced DCPIP form was demonstrated. The oxidized form of DCPIP was shown to be an efficient electron acceptor for terminal iron-sulfur cluster of photosystem I. Likewise methyl viologen, after one-electron reduction, DCPIP transfers an electron to the molecular oxygen. These results were discussed in terms of influence of these interactions on photosystem I reactions with the molecular oxygen and natural electron acceptors.


Assuntos
2,6-Dicloroindofenol/metabolismo , Complexo de Proteína do Fotossistema I/fisiologia , Tetrametilfenilenodiamina/metabolismo , Transporte de Elétrons , Luz , Oxirredução , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Synechocystis/metabolismo
5.
Sci Rep ; 7: 41074, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-28120876

RESUMO

During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.


Assuntos
Coenzimas/química , Coenzimas/metabolismo , Mycobacterium tuberculosis/enzimologia , NAD/química , NAD/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , 2,6-Dicloroindofenol/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Monoterpenos Cicloexânicos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Monoterpenos/metabolismo , Mutagênese Insercional , Oxirredutases/genética , Ligação Proteica , Conformação Proteica , Tiazóis/metabolismo
6.
Appl Microbiol Biotechnol ; 100(2): 697-706, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26452496

RESUMO

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Lignina/metabolismo , Ustilago/enzimologia , 2,6-Dicloroindofenol/metabolismo , Benzoquinonas/metabolismo , Biomassa , Transporte de Elétrons , Oxigênio/metabolismo , Filogenia , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ustilago/metabolismo
7.
Plant Cell Physiol ; 55(7): 1266-75, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24853102

RESUMO

PSII generates strong oxidants used for water oxidation. The secondary electron donor, Y(Z), is Tyr161 on PSII reaction center D1 protein and mediates electron transfer from the oxygen-evolving Mn(4)CaO(5) cluster to the primary electron donor, P680. The latest PSII crystal structure revealed the presence of a hydrogen bond network around Y(Z), which is anticipated to play important roles in the electron and proton transfer reactions. Y(Z) forms a hydrogen bond with His190 which in turn forms a hydrogen bond with Asn298 on D1 protein. Although functional roles of Y(Z) and His190 have already been characterized, little is known about the functional role of Asn298. Here we have generated 19 mutants from a green alga Chlamydomonas reinhardtii, in which the Asn298 has been substituted by each of the other 19 amino acid residues. All mutants showed significantly impaired or no photosynthetic growth. Seven mutants capable of photosynthetic growth showed oxygen-evolving activity although at a significantly reduced rate. Interestingly the oxygen-evolving activity of these mutants was markedly photosensitive. The 19 mutants accumulated PSII at variable levels and showed a light-induced electron transfer reaction from 1,5-diphenylcarbazide (DPC) to 2,6-dichlorophenolindophenol (DCIP), suggesting that Asn298 is important for the function and photoprotection of the Mn(4)CaO(5) cluster.


Assuntos
Asparagina/metabolismo , Chlamydomonas reinhardtii/genética , Oxigênio/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , 2,6-Dicloroindofenol/metabolismo , Substituição de Aminoácidos , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Clorofila/metabolismo , Difenilcarbazida/metabolismo , Transporte de Elétrons , Cinética , Luz , Manganês/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética
8.
Prikl Biokhim Mikrobiol ; 50(6): 570-7, 2014.
Artigo em Russo | MEDLINE | ID: mdl-25726665

RESUMO

Acetic acid bacteria Gluconobacter oxydans subsp. industrius RKM V-1280 were immobilized into a synthetic matrix based on polyvinyl alcohol modified with N-vinylpyrrolidone and used as biocatalysts for the development ofbioanodes for microbial fuel cells. The immobilization method did not significantly affect bacterial substrate specificity. Bioanodes based on immobilized bacteria functioned stably for 7 days. The maximum voltage (fuel cell signal) was reached when 100-130 µM of an electron transport mediator, 2,6-dichlorophenolindophenol, was added into the anode compartment. The fuel cell signals reached a maximum at a glucose concentration higher than 6 mM. The power output of the laboratory model of a fuel cell based on the developed bioanode reached 7 mW/m2 with the use of fermentation industry wastes as fuel.


Assuntos
2,6-Dicloroindofenol/química , Fontes de Energia Bioelétrica , Gluconobacter oxydans/química , Glucose/metabolismo , Polímeros/química , 2,6-Dicloroindofenol/metabolismo , Biocatálise , Células Imobilizadas , Eletrodos , Transporte de Elétrons , Fermentação , Gluconobacter oxydans/metabolismo , Glucose/química , Resíduos Industriais , Oxirredução , Álcool de Polivinil/química , Pirrolidinonas/química
9.
Biotechnol J ; 9(4): 474-82, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24376171

RESUMO

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases - pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, D-amino acid oxidase, and L-lactate oxidase - was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs.


Assuntos
Ensaios Enzimáticos/métodos , Oxirredutases/metabolismo , 2,6-Dicloroindofenol/análise , 2,6-Dicloroindofenol/química , 2,6-Dicloroindofenol/metabolismo , Corantes/análise , Corantes/química , Corantes/metabolismo , Ensaios de Triagem em Larga Escala , Azul de Metileno/análogos & derivados , Azul de Metileno/análise , Azul de Metileno/química , Azul de Metileno/metabolismo , Oxirredução , Fenotiazinas/análise , Fenotiazinas/química , Fenotiazinas/metabolismo
10.
Photosynth Res ; 117(1-3): 385-99, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23794169

RESUMO

Extraction of Ca(2+) from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O2 evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as "the decoupling effect" (Semin et al. Photosynth Res 98:235-249, 2008). Extraction of Cl(-) from Ca(2+)-depleted membranes (PSII[-Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H2O2, and O 2 (·-) , as possible products of water oxidation in PSII(-Ca) membranes was examined. During the investigation of O 2 (·-) production in PSII(-Ca) samples, we found that (i) O 2 (·-) is formed on the acceptor side of PSII due to the reduction of O2; (ii) depletion of Cl(-) does not inhibit water oxidation, but (iii) Cl(-) depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl(-) under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O2, thereby increasing the rate of O 2 (·-) generation. From these observations we conclude that the product of water oxidation is H2O2 and that Cl(-) anions are not involved in the oxidation of water to H2O2 in decoupled PSII(-Ca) membranes. These results also indicate that Cl(-) anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H2O molecules to the Mn4CaO5 cluster and/or facilitate the release of H(+) ions into the lumenal space.


Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Spinacia oleracea/metabolismo , 2,6-Dicloroindofenol/metabolismo , Aminoácidos/metabolismo , Citocromos c/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Radical Hidroxila/metabolismo , Luz , Oxirredução , Oxigênio/metabolismo , Spinacia oleracea/efeitos da radiação , Especificidade por Substrato/efeitos da radiação , Superóxidos/metabolismo , Água/metabolismo
11.
J Allergy Clin Immunol ; 132(3): 696-703.e10, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23683465

RESUMO

BACKGROUND: Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens. OBJECTIVE: We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4. METHODS: Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays. RESULTS: The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein. CONCLUSION: The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs.


Assuntos
Alérgenos/química , Alérgenos/imunologia , Phleum/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Pólen/imunologia , 2,6-Dicloroindofenol/metabolismo , Alérgenos/metabolismo , Sequência de Aminoácidos , Basófilos/imunologia , Glucose 1-Desidrogenase/química , Glucose 1-Desidrogenase/imunologia , Glucose 1-Desidrogenase/metabolismo , Imunoglobulina E/sangue , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Pólen/química , Conformação Proteica , Proteínas Recombinantes/imunologia
12.
Appl Microbiol Biotechnol ; 97(14): 6397-412, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23519735

RESUMO

Gluconobacter oxydans, like all acetic acid bacteria, has several membrane-bound dehydrogenases, which oxidize a multitude of alcohols and polyols in a stereo- and regio-selective manner. Many membrane-bound dehydrogenases have been purified from various acetic acid bacteria, but in most cases without reporting associated sequence information. We constructed clean deletions of all membrane-bound dehydrogenases in G. oxydans 621H and investigated the resulting changes in carbon utilization and physiology of the organism during growth on fructose, mannitol, and glucose. Furthermore, we studied the substrate oxidation spectra of a set of strains where the membrane-bound dehydrogenases were consecutively deleted using a newly developed whole-cell 2,6-dichlorophenolindophenol (DCPIP) activity assay in microtiter plates. This allowed a detailed and comprehensive in vivo characterization of each membrane-bound dehydrogenase in terms of substrate specificity. The assays revealed that general rules can be established for some of the enzymes and extended the known substrate spectra of some enzymes. It was also possible to assign proteins whose purification and characterization had been reported previously, to their corresponding genes. Our data demonstrate that there are less membrane-bound dehydrogenases in G. oxydans 621H than expected and that the deletion of all of them is not lethal for the organism.


Assuntos
Proteínas de Bactérias/genética , Membrana Celular/enzimologia , Deleção de Genes , Gluconobacter oxydans/enzimologia , Oxirredutases/genética , 2,6-Dicloroindofenol/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Membrana Celular/química , Membrana Celular/genética , Gluconobacter oxydans/genética , Gluconobacter oxydans/crescimento & desenvolvimento , Oxirredutases/química , Oxirredutases/metabolismo , Especificidade por Substrato
13.
Appl Microbiol Biotechnol ; 97(8): 3419-27, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22752365

RESUMO

Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4ß4 and αßγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the ß subunit (PDHß) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHßs from α4ß4- and αßγδ-type enzyme complexes, eight PDHßs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4ß4-type PDHßs showed greater thermostability than most of the αßγδ-type PDHßs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4ß4-type PDHßs utilized ferricyanide as the most preferable electron acceptor, whereas αßγδ-type PDHßs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHßs likely reflect whether they were from an αßγδ- or α4ß4-type complex, though the wider divergence observed within αßγδ-type PDHßs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHßs did not reflect the complex type. Interestingly, the k cat value for free α4ß4-type PDHß from P. horikoshii was much larger than the value for the same subunit within the α4ß4-complex. This indicates that the isolated PDHß could be a useful element for an electrochemical system for detection of L-proline.


Assuntos
Prolina Oxidase/metabolismo , Prolina/metabolismo , Pyrococcus/enzimologia , Thermococcus/enzimologia , 2,6-Dicloroindofenol/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Ferricianetos/metabolismo , Expressão Gênica , Temperatura Alta , Cinética , Prolina Oxidase/genética , Estabilidade Proteica , Pyrococcus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Thermococcus/genética , Fatores de Tempo
14.
Anal Biochem ; 419(1): 26-32, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21864496

RESUMO

Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S.cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.


Assuntos
2,6-Dicloroindofenol/metabolismo , Candida albicans/crescimento & desenvolvimento , Viabilidade Microbiana , NAD/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida glabrata/crescimento & desenvolvimento , Candida glabrata/metabolismo , Catálise , Cor , Eletroquímica , Transporte de Elétrons/efeitos dos fármacos , Ativação Enzimática , Proteínas Fúngicas , Galactose/metabolismo , Glucose/metabolismo , Técnicas Microbiológicas , NADH Desidrogenase/metabolismo , Oxirredução , Oxigênio/metabolismo , Rotenona/farmacologia , Saccharomyces cerevisiae/metabolismo , Espectrofotometria
15.
Appl Biochem Biotechnol ; 163(3): 404-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20936374

RESUMO

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k (ox) = 660 M(-1) s(-1), while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.


Assuntos
Acinetobacter/enzimologia , Desidrogenases de Carboidrato/metabolismo , Elétrons , Escherichia coli/enzimologia , Glucose Desidrogenase/metabolismo , 2,6-Dicloroindofenol/metabolismo , Biocatálise , Cinética , Oxirredução , Oxigênio/metabolismo , Propionatos/metabolismo , Especificidade por Substrato , Trissacarídeos/metabolismo
16.
Antimicrob Agents Chemother ; 54(10): 4335-42, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20625147

RESUMO

Two bacterial species with different metabolic features, namely, Pseudomonas aeruginosa and Lactococcus lactis, were used as a comparative experimental model to investigate the antimicrobial target and mechanism of transferrins. In anaerobiosis, P. aeruginosa cells were not susceptible to lactoferrin (hLf) or transferrin (hTf). In aerobiosis, the cells were susceptible but O(2) consumption was not modified, indicating that components of the electron transport chain (ETC) were not targeted. However, the respiratory chain inhibitor piericidin A significantly reduced the killing activity of both proteins. Moreover, 2,6-dichlorophenolindophenol (DCIP), a reducing agent that accepts electrons from the ETC coupled to H(+) extrusion, made P. aeruginosa susceptible to hLf and hTf in anaerobiosis. These results indicated that active cooperation of the cell was indispensable for the antimicrobial effect. For L. lactis cells lacking an ETC, the absence of a detectable transmembrane electrical potential in hLf-treated cells suggested a loss of H(+)-ATPase activity. Furthermore, the inhibition of ATPase activity and H(+) translocation (inverted membrane vesicles) provided direct evidence of the ability of hLf to inhibit H(+)-ATPase in L. lactis. Based on these data, we propose that hLf and hTf also inhibit the H(+)-ATPase of respiring P. aeruginosa cells. Such inhibition thereby interferes with reentry of H(+) from the periplasmic space to the cytoplasm, resulting in perturbation of intracellular pH and the transmembrane proton gradient. Consistent with this hypothesis, periplasmic H(+) accumulation was prevented by anaerobiosis or by piericidin A or was induced by DCIP in anaerobiosis. Collectively, these results indicate that transferrins target H(+)-ATPase and interfere with H(+) translocation, yielding a lethal effect in vitro.


Assuntos
Lactococcus lactis/efeitos dos fármacos , Lactococcus lactis/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Transferrina/farmacologia , 2,6-Dicloroindofenol/metabolismo , Aerobiose , Anaerobiose , Anti-Infecciosos/farmacologia , Lactoferrina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo
17.
J Bacteriol ; 192(16): 4172-80, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20562311

RESUMO

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.


Assuntos
Cobre/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/fisiologia , Nitrorredutases/genética , Nitrorredutases/metabolismo , Estresse Oxidativo , Estresse Fisiológico , 2,6-Dicloroindofenol/metabolismo , 4-Nitroquinolina-1-Óxido/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cádmio/metabolismo , Catalase/química , Catalase/genética , Catalase/isolamento & purificação , Catalase/metabolismo , Cristalografia por Raios X , Flavoproteínas/química , Flavoproteínas/genética , Flavoproteínas/isolamento & purificação , Flavoproteínas/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Cinética , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Nitrorredutases/química , Nitrorredutases/isolamento & purificação , Oxidantes/metabolismo , Oxirredução , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Prata/metabolismo
18.
Phytochemistry ; 71(1): 27-35, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19883924

RESUMO

Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including biosynthesis of secondary metabolites.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Expressão Gênica , Gossypium/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Plantas/metabolismo , 2,6-Dicloroindofenol/metabolismo , Sequência de Aminoácidos , Citocromos c/metabolismo , DNA Complementar , Ferricianetos/metabolismo , Genes de Plantas , Gossypium/genética , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , Filogenia , Doenças das Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes , Homologia de Sequência de Aminoácidos , Transcinamato 4-Mono-Oxigenase/metabolismo
19.
Photosynth Res ; 98(1-3): 235-49, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18814052

RESUMO

Extraction of Ca(2+) from the O(2)-evolving complex (OEC) of photosystem II (PSII) membranes with 2 M NaCl in the light (PSII(-Ca/NaCl)) results in 90% inhibition of the O(2)-evolution reaction. However, electron transfer from the donor to acceptor side of PSII, measured as the reduction of the exogenous acceptor 2,6-dichlorophenolindophenol (DCIP) under continuous light, is inhibited by only 30%. Thus, calcium extraction from the OEC inhibits the synthesis of molecular O(2) but not the oxidation of a substrate we term X, the source of electrons for DCIP reduction. The presence of electron transfer across PSII(-Ca/NaCl) membranes was demonstrated using fluorescence induction kinetics, a method that does not require an artificial acceptor. The calcium chelator, EGTA (5 mM), when added to PSII(-Ca/NaCl) membranes, does not affect the inhibition of O(2) evolution by NaCl but does inhibit DCIP reduction up to 92% (the reason why electron transport in Ca(2+)-depleted materials has not been noticed before). Another chelator, sodium citrate (citrate/low pH method of calcium extraction), also inhibits both O(2) evolution and DCIP reduction. The role of all buffer components (including bicarbonate and sucrose) as possible sources of electrons for PSII(-Ca/NaCl) membranes was investigated, but only the absence of chloride anions strongly inhibited the rate of DCIP reduction. Substitution of other anions for chloride indicates that Cl(-) serves its well-known role as an OEC cofactor, but it is not substrate X. Multiple turnover flash experiments have shown a period of four oscillations of the fluorescence yield (both the maximum level, F(max), and the fluorescence level measured 50 s after an actinic flash in the presence of DCMU) in native PSII membranes, reflecting the normal function of the OEC, but the absence of oscillations in PSII(-Ca/NaCl) samples. Thus, PSII(-Ca/NaCl) samples do not evolve O(2) but do transfer electrons from the donor to acceptor sides and exhibit a disrupted S-state cycle. We explain these results as follows. In Ca(2+)-depleted PSII membranes, obtained without chelators, the oxidation of the OEC stops after the absorption of three quanta of light (from the S1 state), which should convert the native OEC to the S4 state. An one-electron oxidation of the water molecule bound to the Mn cluster then occurs (the second substrate water molecule is absent due to the absence of calcium), and the OEC returns to the S3 state. The appearance of a sub-cycle within the S-state cycle between S3-like and S4-like states supplies electrons (substrate X is postulated to be OH(-)), explains the absence of O(2) production, and results in the absence of a period of four oscillation of the normal functional parameters, such as the fluorescence yield or the EPR signal from S2. Chloride anions probably keep the redox potential of the Mn cluster low enough for its oxidation by Y(Z)(*).


Assuntos
Cálcio/metabolismo , Transporte de Elétrons , Oxigênio/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , 2,6-Dicloroindofenol/metabolismo , Quelantes/metabolismo , Cloretos/metabolismo , Fluorescência , Manganês/metabolismo , Spinacia oleracea
20.
Anal Chem ; 80(3): 843-8, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18181646

RESUMO

Screen-printed carbon electrode arrays were treated to allow respiratory activity-based measurement of antibiotic susceptibility with Escherichia coli JM105. Carbon working electrodes were examined for antibiotic adsorption and were pretreated with various electrochemical and chemical protocols to minimize antibiotic adsorption. Treatment by voltammetry in basic solution or by chemical modification with poly-L-lysine or chitosan were found to be effective methods for the elimination of adsorption of the studied group of 17 antibiotics, which comprised several classes and modes of action. Measurements consisted of two-electrode amperometry of the bacterial suspension after 10 min of incubation with antibiotic followed by addition of an oxidative cocktail of ferricyanide and dichlorophenolindophenol for a further 10 min; response currents, which indicate the extent of reduction of ferricyanide to ferrocyanide by cellular respiratory activity, decrease with increasing concentration of antibiotic present in the initial 10 min incubation. IC50 values obtained for chloramphenicol with these electrode modification methods are consistent at 2.0 +/- 0.2 mM, in approximate agreement with previously reported respiration-based results for this organism but significantly higher than values reported for growth-based antibiotic susceptibility testing methods.


Assuntos
Antibacterianos/farmacologia , Técnicas Bacteriológicas/métodos , Técnicas Biossensoriais/métodos , Carbono/química , Escherichia coli/efeitos dos fármacos , 2,6-Dicloroindofenol/metabolismo , Adsorção , Técnicas Bacteriológicas/instrumentação , Técnicas Biossensoriais/instrumentação , Quitosana/química , Cloranfenicol/metabolismo , Eletroquímica , Eletrodos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Ferricianetos/metabolismo , Ferrocianetos/metabolismo , Testes de Sensibilidade Microbiana , Oxirredução , Polilisina/química , Fatores de Tempo
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