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‣ Tratamento de vinhaça por processos físico-químicos de precipitação química e flotação e sua utilização como meio de cultivo para a microalga de potencial bioenergético, Chlorella vulgaris; Stillage treatment by physical-chemical processes such as chemical precipitation and flotation and its use as a culture medium for the bioenergetic potentially microalgae, Chlorella vulgaris

Bichara, Andressa
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 24/10/2014 Português
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A vinhaça é um dos resíduos provenientes da indústria sucroalcooleira, sendo gerados em média 12 litros do efluente para cada litro de álcool produzido. Uma elevada carga orgânica torna a vinhaça potencial agente poluidor, podendo a disposição direta no solo representar solução ambientalmente desvantajosa para este resíduo. Diante do crescimento do setor sucroalcooleiro no Brasil e da necessidade de gestão do volumoso resíduo, este estudo avaliou duas formas de tratamentos físico-químicos da vinhaça, a precipitação química e a flotação por ar dissolvido (FAD), além de um tratamento biológico através do cultivo de microalgas no efluente do sistema FAD. Avaliou-se, adicionalmente, o potencial nutritivo do sólido separado pela FAD para aplicação como suprimento em alimentação animal. A precipitação química foi estudada variando-se pH (via adição NaOH) e tempo de reação, tendo atingido eficiências de remoção de fósforo, amônia e potássio de 31,3 ± 3,4%, 7,8 ± 4,1% e 9,7 ± 0,7%, respectivamente na faixa de pH de 8,5 a 9,5. Na FAD apenas o parâmetro dosagem apresentou significância estatística na separação dos sólidos suspensos da vinhaça. A dosagem e o percentual de recirculação foram avaliados em ensaios de clarificação e de espessamento de lodo de vinhaça. Desses ensaios obteve-se que todas as faixas de dosagens e recirculações geraram eficiências de remoção de sólidos extremamente elevadas...

‣ Flotação por Ar Dissolvido (FAD) de micropartículas, caracterização de microbolhas e medidas de força de interação bolha-partícula

Englert, Alexandre Hahn
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Tese de Doutorado Formato: application/pdf
Português
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O presente trabalho descreve um estudo teórico-experimental da flotação por ar dissolvido (FAD) de micropartículas (diâmetro dp < 100 μm), incluindo caracterização de microbolhas (db < 100 μm) pela técnica LTM-BSizer de determinação da distribuição de tamanho de bolhas (DTB) e medidas diretas da força de interação bolha-partícula via microscopia de força atômica (AFM). Visando aperfeiçoar a técnica LTM-BSizer, foi desenvolvido um novo procedimento (NP) automático de processamento/análise de imagens de bolhas. O NP consistiu em um algoritmo contendo as etapas de conversão das imagens coloridas para tons de cinza, limiarização, preenchimento dos “buracos” das bolhas e análise. Imagens de microbolhas geradas por uma bomba centrífuga multiestágio foram utilizadas para comparar os resultados obtidos pelo NP com os obtidos pelo antigo procedimento (AP) da técnica. Diferenças menores que 20 % foram observadas nos resultados obtidos pelos dois procedimentos para o diâmetro médio aritmético, desvio-padrão e diâmetro médio de Sauter das bolhas. O processamento/análise com o NP foi realizado em um tempo inferior (> 30 %) ao requerido pelo AP, mostrando ainda a vantagem de detectar confiavelmente bolhas circulares não-agrupadas nas imagens. Estudos experimentais de FAD de micropartículas de quartzo (diâmetro médio volumétrico de 13...

‣ Estudos de Flotação por Ar Dissolvido com Bomba Multifásica (FAD-B) e Sedimentação Lamelar (SL) no tratamento de água bruta para abastecimento público (Canoas-RS)

Azevedo, André Camargo de
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
Português
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Foi realizado um estudo experimental, em nível piloto, comparativo entre os processos de Flotação por Ar Dissolvido com Bomba Multifásica (FAD-B) e Sedimentação Lamelar (SL) no tratamento de água bruta para abastecimento público do município de Canoas-RS. O parâmetro de avaliação desses processos de separação sólido-líquido foi a redução de turbidez de água bruta do Arroio das Garças, da ETA Rio Branco, operada pela Companhia Riograndense de Saneamento (Corsan). Em nível de bancada, foram determinados e otimizados os parâmetros químicos e físico-químicos de agregação (pH do meio, concentração de reagentes e gradientes de velocidade de mistura rápida e lenta) e parâmetros de operação da flotação (taxa de reciclo e pressão de saturação). Os melhores resultados, em nível de bancada, de Coagulação-Flotação FAD foram obtidos em pH na faixa de 6.1 – 6.3, concentração de sulfato de alumínio igual a 30 mg.L-1, valores de G de mistura rápida entre 700 e 1300 s-1, G de mistura lenta igual 80 s-1, 20 % de taxa de reciclo e 4 atm de pressão de saturação de água com ar. A turbidez residual média foi de aproximadamente 2,8 NTU ou 94 % de redução de turbidez inicial. Ainda foi avaliado o efeito do uso de três tipos de polímeros floculantes após coagulação com sulfato de alumínio...

‣ The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase

Pedrolli, Danielle B.; Nakanishi, Shinobu; Barile, Maria; Mansurova, Madina; Carmona, Eleonora C.; Lux, Andreas; Gaertner, Wolfgang; Mack, Matthias
Fonte: Pergamon-Elsevier B.V. Ltd Publicador: Pergamon-Elsevier B.V. Ltd
Tipo: Artigo de Revista Científica Formato: 1853-1859
Português
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); The non-pathogenic Gram-positive soil bacterium Streptomyces davawensis synthesizes the riboflavin (vitamin B(2)) analogs roseoflavin (RoF) and 8-demethyl-8-amino-riboflavin (AF). Both compounds are antibiotics. Notably, a number of other riboflavin analogs are currently under investigation with regard to the development of novel antiinfectives. As a first step towards understanding the metabolism of riboflavin analogs in humans, the key enzymes flavokinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) were studied. Human flavokinase efficiently converted RoF and AF to roseoflavin mononucleotide (RoFMN) and 8-demethyl-8-amino-riboflavin mononucleotide (AFMN), respectively. Human FAD synthetase accepted RoFMN but not AFMN as a substrate. Consequently, roseoflavin adenine dinucleotide (RoFAD) was synthesized by the latter enzyme but not 8-demethyl-8-amino-riboflavin adenine dinucleotide (AFAD). The cofactor analogs RoFMN, AFMN and RoFAD have different physicochemical properties as compared to FMN and FAD. Thus, the cofactor analogs have the potential to render flavoenzymes inactive, which may negatively affect human metabolism. RoF, but not AF, was found to inhibit human flavokinase. In summary...

‣ Clarificação por flotação com ar dissolvido (FAD) da calda de açúcar cristal para produção de açúcar refinado

Crema, Leandra Cristina
Fonte: Universidade Estadual Paulista (UNESP) Publicador: Universidade Estadual Paulista (UNESP)
Tipo: Dissertação de Mestrado Formato: 128 f. : il.
Português
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE; O Brasil é o maior produtor mundial de açúcar de cana o qual apresenta grande importância para o crescimento do País. Uma das etapas mais importantes na produção de açúcar refinado é a etapa de clarificação da calda de açúcar, a partir da dissolução do açúcar cristal bruto (Demerara, VHP ou V-VHP), pois influencia as etapas subsequentes do processo para obtenção de produto de boa qualidade. No Brasil, geralmente nos processos convencionais de produção de açúcar, as usinas e refinarias utilizam a sulfitação para clarificar o caldo e xarope de cana, que emprega o dióxido de enxofre (SO2 ), a qual tem sido muito questionada por gerar problemas operacionais, tecnológicos, ambientais e restrições nas normas de segurança alimentar. Neste sentido, existe uma forte demanda por pesquisas que visem o desenvolvimento de tecnologias que busquem um melho r rendimento, qualidade do produto e aproveitamento dos recursos naturais existentes. Entre essas tecnologias, o processo de flotação com ar dissolvido (FAD) utilizando um polímero catiônico para a neutralização e coagulação de partículas coloidais e em suspensão...

‣ Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

Segal, A W; West, I; Wientjes, F; Nugent, J H; Chavan, A J; Haley, B; Garcia, R C; Rosen, H; Scrace, G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1992 Português
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The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome...

‣ Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

Weyler, W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1989 Português
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I present the first clear evidence that the protein: FAD ratio in human monoamine oxidase A and bovine monoamine oxidase B has an upper limit of 65 kDa and 57 kDa per FAD, respectively. To now it had been assumed that the protein: FAD ratio was 100-120 kDa to 1 FAD and that there was one FAD per two subunits which were assumed to be of the same size. For the present work the purity of monoamine oxidase A and monoamine oxidase B was improved over that previously achieved. Protein was determined by quantitative amino acid analysis and FAD content was measured by spectrophotometric titration of SDS-denatured enzyme with NaS2O4 standardized against riboflavin. The cause of the previous misassignment of the protein: FAD ratio was judged as having been due to the use of impure enzyme preparations. Knowledge of the correct protein: FAD ratio is important in devising cloning strategies for this enzyme, in understanding its structure, function, mechanism, and in the studies of its biosynthesis.

‣ The association of FAD with the cytochrome b−245 of human neutrophils

Cross, Andrew R.; Jones, Owen T. G.; Garcia, Rudolfo; Segal, Anthony W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/12/1982 Português
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A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b−245 at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b−245 the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b−245 and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1...

‣ Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride.

Covès, J; Lebrun, C; Gervasi, G; Dalbon, P; Fontecave, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/09/1999 Português
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SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized. It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor. Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)). In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD. However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR [Tew (1993) Biochemistry 32, 10209-10215]. Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein. Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

‣ A fraction (FAd) from Trypanosoma cruzi epimastigotes depresses the immune response in mice.

Corsini, A C; Costa, M G; Oliveira, O L; Camargo, I J; Rangel, H A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1980 Português
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The primary immune response to SRBC in BALB/c mice was depressed when they were injected with a fraction (FAd) obtained from Trypanosoma cruzi epimastigotes grown in LIT medium. Plaque-forming cell (PFC) number was 50% less than controls when FAd was injected i.v. 15 min before antigen in doses ranging from 70 microgram up to 400 microgram of protein. Similar depression was observed when 100 microgram FAd was injected up to 6 h before antigen. There was no shift in the peak response to SRBC, neither was depression detected, when a total of 100 microgram FAd protein was given in 20 microgram amounts twice a day before immunization. Mice injected with FAd fraction only showed no increase in background PFC. Both secondary IgM and secondary IgG PFC were depressed when FAd was given before the boosting injection. However, only IgG PFC were depressed when FAd was injected before the priming dose. The delayed-type hypersensitivity reaction to DNFB was depressed when animals were injected either during the 3 days after sensitization or with a single dose of 100 microgram of protein of FAd on day of challenge. Bone marrow colony-forming units in spleens of mice injected with FAd were depressed and nodules in the treated animals were smaller than in controls. We conclude that FAd affects humoral and cell-mediated immune responses by interfering with cell division at some stage of the cell cycle.

‣ Chromosome 14 and late-onset familial Alzheimer disease (FAD)

Schellenberg, Gerard D.; Payami, Haydeh; Wijsman, Ellen M.; Orr, Harry T.; Goddard, Katrina A. B.; Anderson, Leojean; Nemens, Ellen; White, June A.; Alonso, M. Elisa; Ball, Melvyn J.; Kaye, Jeffrey; Morris, John C.; Chui, Helena; Sadovnick, A. Dessa; Hest
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1993 Português
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Familial Alzheimer disease (FAD) is genetically heterogeneous. Two loci responsible for early-onset FAD have been identified: the amyloid precursor protein gene on chromosome 21 and the as-yet-unidentified locus on chromosome 14. The genetics of late-onset FAD is unresolved. Maximum-likelihood, affected-pedigree-member (APM), and sib-pair analyses were used, in 49 families with a mean age at onset ≥60 years, to determine whether the chromosome 14 locus is responsible for late-onset FAD. The markers used were D14S53, D14S43, and D14S52. The LOD score method was used to test for linkage of late-onset FAD to the chromosome 14 markers, under three different models: age-dependent penetrance, an affected-only analysis, and age-dependent penetrance with allowance for possible age-dependent sporadic cases. No evidence for linkage was obtained under any of these conditions for the late-onset kindreds, and strong evidence against linkage (LOD score ≤ –2.0) to this region was obtained. Heterogeneity tests of the LOD score results for the combined group of families (early onset, Volga Germans, and late onset) favored the hypothesis of linkage to chromosome 14 with genetic heterogeneity. The positive results are primarily from early-onset families. APM analysis gave significant evidence for linkage of D14S43 and D14S52 to FAD in early-onset kindreds (P < .02). No evidence for linkage was found for the entire late-onset family group. Significant evidence for linkage to D14S52...

‣ Specific stimulation of peripheral blood mononuclear cells from patients with acute myocarditis by peptide-bound flavin adenine dinucleotide (FAD), a naturally occurring autologous hapten

CICEK, G; SCHILTZ, E; STAIGER, J; NEUMANN, F-J; MELCHERS, I; BRANDSCH, R
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /05/2003 Português
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The tryptic FAD-peptide carrying the flavin in 8α-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-γ]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten...

‣ Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.

van Berkel, W. J.; Eppink, M. H.; Schreuder, H. A.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /12/1994 Português
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The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3...

‣ ADP Competes with FAD Binding in Putrescine Oxidase*

van Hellemond, Erik W.; Mazon, Hortense; Heck, Albert J.; van den Heuvel, Robert H. H.; Heuts, Dominic P. H. M.; Janssen, Dick B.; Fraaije, Marco W.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 17/10/2008 Português
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Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuORh) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuORh uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuORh that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuORh A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.

‣ Flavin Nucleotide Metabolism in Plants: MONOFUNCTIONAL ENZYMES SYNTHESIZE FAD IN PLASTIDS*

Sandoval, Francisco J.; Zhang, Yi; Roje, Sanja
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 07/11/2008 Português
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FAD synthetases (EC 2.7.7.2) catalyze biosynthesis of FAD from FMN and ATP. Monofunctional FAD synthetases are known to exist in mammals and yeast; bifunctional enzymes also catalyzing phosphorylation of riboflavin to FMN are known to exist in bacteria. Previously known eukaryotic enzymes with FAD synthetase activity have no sequence similarity to prokaryotic enzymes with riboflavin kinase and FAD synthetase activities. Proteins homologous to bacterial bifunctional FAD synthetases, yet shorter and lacking amino acid motifs at the C terminus, were found by bioinformatic analyses in vascular plant genomes, suggesting that plants contain a type of FAD synthetase previously known to exist only in prokaryotes. The Arabidopsis thaliana genome encodes two of such proteins. Both proteins, which we named AtRibF1 and AtRibF2, carry N-terminal extensions with characteristics of organellar targeting peptides. AtRibF1 and AtRibF2 cDNAs were cloned by reverse transcription-PCR. Only FAD synthetase activity was detected in the recombinant enzymes produced in Escherichia coli. FMN and ATP inhibited both enzymes. Kinetic parameters of AtRibF1 and AtRibF2 for the two substrates were similar. Confocal microscopy of protoplasts transformed with enhanced green fluorescence protein-fused proteins showed that AtRibF1 and AtRibF2 are targeted to plastids. In agreement with subcellular localization to plastids...

‣ Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

Debeurme, Franck; Picciocchi, Antoine; Dagher, Marie-Claire; Grunwald, Didier; Beaumel, Sylvain; Fieschi, Franck; Stasia, Marie-José
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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The X+-linked chronic granulomatous disease (X+-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X+-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X+-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However...

‣ Fusobacterium nucleatum-associated β-Defensin Inducer (FAD-I): IDENTIFICATION, ISOLATION, AND FUNCTIONAL EVALUATION*

Gupta, Sanhita; Ghosh, Santosh K.; Scott, Mary E.; Bainbridge, Brian; Jiang, Bin; Lamont, Richard J.; McCormick, Thomas S.; Weinberg, Aaron
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Human β-defensins (hBDs) are small, cationic antimicrobial peptides, secreted by mucosal epithelial cells that regulate adaptive immune functions. We previously reported that Fusobacterium nucleatum, a ubiquitous Gram-negative bacterium of the human oral cavity, induces human β-defensin 2 (hBD2) upon contact with primary oral epithelial cells. We now report the isolation and characterization of an F. nucleatum (ATCC 25586)-associated defensin inducer (FAD-I). Biochemical approaches revealed a cell wall fraction containing four proteins that stimulated the production of hBD2 in human oral epithelial cells (HOECs). Cross-referencing of the N-terminal sequences of these proteins with the F. nucleatum genome revealed that the genes encoding the proteins were FadA, FN1527, FN1529, and FN1792. Quantitative PCR of HOEC monolayers challenged with Escherichia coli clones expressing the respective cell wall proteins revealed that FN1527 was most active in the induction of hBD2 and hence was termed FAD-I. We tagged FN1527 with a c-myc epitope on the C-terminal end to identify and purify it from the E. coli clone. Purified FN1527 (FAD-I) induced hBD2 mRNA and protein expression in HOEC monolayers. F. nucleatum cell wall and FAD-I induced hBD2 via TLR2. Porphorymonas gingivalis...

‣ FAD Binding by ApbE Protein from Salmonella enterica: a New Class of FAD-Binding Proteins▿ †

Boyd, Jeffery M.; Endrizzi, James A.; Hamilton, Trinity L.; Christopherson, Melissa R.; Mulder, David W.; Downs, Diana M.; Peters, John W.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
27.338323%
The periplasmic protein ApbE was identified through the analysis of several mutants defective in thiamine biosynthesis and was implicated as having a role in iron-sulfur cluster biosynthesis or repair. While mutations in apbE cause decreased activity of several iron-sulfur enzymes in vivo, the specific role of ApbE remains unknown. Members of the AbpE family include NosX and RnfF, which have been implicated in oxidation-reduction associated with nitrous oxide and nitrogen metabolism, respectively. In this work, we show that ApbE binds one FAD molecule per monomeric unit. The structure of ApbE in the presence of bound FAD reveals a new FAD-binding motif. Protein variants that are nonfunctional in vivo were generated by random and targeted mutagenesis. Each variant was substituted in the environment of the FAD and analyzed for FAD binding after reconstitution. The variant that altered a key tyrosine residue involved in FAD binding prevented reconstitution of the protein.

‣ Bacterial Over-Expression and Purification of the 3′phosphoadenosine 5′phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

Miccolis, Angelica; Galluccio, Michele; Giancaspero, Teresa Anna; Indiveri, Cesare; Barile, Maria
Fonte: Molecular Diversity Preservation International (MDPI) Publicador: Molecular Diversity Preservation International (MDPI)
Tipo: Artigo de Revista Científica
Publicado em 11/12/2012 Português
Relevância na Pesquisa
27.29555%
FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ1-328-hFADS enzyme shows a slight, but not significant, change of Km values (0.24 and 6.23 μM for FMN and ATP, respectively) and of kcat (4.2 × 10−2 s−1) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ1-328-hFADS versus wild-type protein.

‣ Sequence-structure analysis of FAD-containing proteins

Dym, Orly; Eisenberg, David
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/2001 Português
Relevância na Pesquisa
27.44461%
We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless...