RESUMO
Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.
Assuntos
Coenzimas/metabolismo , L-Iditol 2-Desidrogenase/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Prunus persica/enzimologia , Cinética , L-Iditol 2-Desidrogenase/química , L-Iditol 2-Desidrogenase/genética , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMO
Many cities worldwide are established adjacent to estuaries and their catchments resulting in estuarine contamination due to intense anthropogenic activities. The aim of this study was to evaluate if fish living in an estuarine urban waterway were affected by contamination, via the measurement of a suite of biomarkers of fish health. Black bream (Acanthopagrus butcheri) were sampled in a small urban embayment and a suite of biomarkers of fish health measured. These were condition factor (CF), liver somatic index (LSI), gonadosomatic index (GSI), hepatic EROD activity, polycyclic aromatic hydrocarbon (PAH) biliary metabolites, serum sorbitol dehydrogenase (s-SDH) and branchial enzymes cytochrome C oxidase (CCO), and lactate dehydrogenase (LDH) activities. The biomarkers of exposure EROD activity, and pyrene- and B(a)P-type biliary metabolites confirmed current or recent exposure of the fish and that fish were metabolizing contaminants. Relative to a reference site, LSI was higher in fish collected in the urban inlet as was the metabolic enzyme LDH activity. CF, GSI, s-SDH, CCO, and naphthalene-type metabolites were at similar levels in the urban inlet relative to the reference site. PAH biliary metabolite ratios of high-molecular-weight to low-molecular-weight suggest that fish from the urban inlet were exposed to pyrogenic PAHs, likely from legacy contamination and road runoff entering the embayment. Similarly, the sediment PAH ratios and the freshness indices suggested legacy contamination of a pyrogenic source, likely originating from the adjacent historic gasworks site and a degree of contamination of petrogenic nature entering the inlet via storm water discharge. Biomarkers of exposure and effect confirmed that black bream collected in the Claisebrook Cove inlet, Western Australia, are currently exposed to contamination and are experiencing metabolic perturbations not observed in fish collected at a nearby reference site.
Assuntos
Perciformes/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Baías , Bile/metabolismo , Biomarcadores/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Monitoramento Ambiental , Estuários , L-Iditol 2-Desidrogenase/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismoRESUMO
The increment in crude oil exploitation over the last decades has considerably increased the risk of polycyclic aromatic hydrocarbon (PAH) contamination to Amazonian aquatic environments, especially for the black water environments such as the Rio Negro. The present work was designed to evaluate the acute toxicity of the Urucu crude oil (CO), the chemically dispersed Urucu crude oil (CO + D), and the dispersant alone (D) to the Amazonian fish Colossoma macropomum. Acute toxicity tests were performed, using a more realistic approach, where fish were acclimated to both groundwater (GW), used as internal control, and natural Rio Negro water (RNW) and exposed to CO, CO + D and D. Then, biomarkers such as ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), lipid peroxidation (LPO), serum sorbitol dehydrogenase (s-SDH) in liver, DNA damage in blood cells, and the presence of the benzo[a]pyrene-type, pyrene-type, and naphthalene-type metabolites in fish bile were assessed. Fish exposed to CO and CO + D, at both water types tested, presented increased biomarker responses and higher PAH-type metabolites in the bile. However, fish exposed to these treatments after the acclimation to RNW increased the levels of LPO, s-SDH (hepatotoxicity), DNA damage in blood cells (genotoxicity), and benzo[a]pyrene-type metabolites when compared to fish in GW. Our data suggests that some physicochemical properties of Rio Negro water (i.e., presence of natural organic matter (NOM)) might cause mild chemical stress responses in fish, which can make it more susceptible to oxidative stress following exposure to crude oil, particularly to those chemically dispersed.
Assuntos
Peixes/metabolismo , Petróleo/toxicidade , Rios/química , Poluentes Químicos da Água/toxicidade , Animais , Bile/química , Biomarcadores/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Dano ao DNA , Proteínas de Peixes/metabolismo , L-Iditol 2-Desidrogenase/metabolismo , Peroxidação de Lipídeos , Fígado/metabolismo , Superóxido Dismutase/metabolismo , Testes de Toxicidade AgudaRESUMO
Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought.
Assuntos
Arabidopsis/enzimologia , L-Iditol 2-Desidrogenase/metabolismo , Sorbitol/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Citosol/enzimologia , Desidratação , Flores/enzimologia , Flores/crescimento & desenvolvimento , Flores/ultraestrutura , Cinética , L-Iditol 2-Desidrogenase/genética , Dados de Sequência Molecular , Mutação , NAD/metabolismo , Especificidade de Órgãos , Fenótipo , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/ultraestrutura , Caules de Planta/enzimologia , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/ultraestrutura , Proteínas Recombinantes de Fusão , Ribitol/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Sementes/ultraestrutura , Alinhamento de Sequência , Xilitol/metabolismoRESUMO
Twenty-eight Candida albicans strains obtained from women with vaginal candidiasis were tested for phospholipase and proteinase production and clustered by multilocus enzyme electrophoresis (MLEE). The proteolytic and phospholipidic activity were considered moderate (0.56 ± 0.12 mm and 0.53 ± 0.09 mm, respectively) for all isolates. The isoenzymes malate dehydrogenase (MDH) and sorbitol dehydrogenase (SDH) showed strong intra-specific discriminatory power. The numerical and genetic interpretation of the bands produced by the isoenzymes tested presented similar discriminatory power. The genetic diversity of the isolates was measured by allelic and genic frequency, perceptual index of polymorphic loci (P = 87.5%), average number of alleles per locus, average number of alleles per polymorphic locus, average heterozygosity observed and average heterozygosity expected. We verified that three isoenzymatic loci (Adh, Gdh and Sdh-2) were not in Hardy-Weinberg equilibrium. A dendrogram constructed based on the genetic distance matrix of Nei showed seven clusters; 57.15% (16) of the isolates were considered highly related or indistinguishable, and 42.85% were considered moderately related or unrelated. We did not find a relationship between the clusters and the exoenzymes production.
Assuntos
Candida albicans/enzimologia , Candidíase Vulvovaginal/microbiologia , Eletroforese em Gel de Poliacrilamida/métodos , Adolescente , Adulto , Alelos , Candida albicans/genética , Candida albicans/isolamento & purificação , Análise por Conglomerados , Ativação Enzimática , Ensaios Enzimáticos/métodos , Feminino , Frequência do Gene , Loci Gênicos , Variação Genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Iditol 2-Desidrogenase/genética , L-Iditol 2-Desidrogenase/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Fenótipo , Fosfolipases/genética , Fosfolipases/metabolismo , Adulto JovemRESUMO
The first step in sucrose use by maize kernels produces fructose, regardless of whether the initial reaction is catalyzed by an invertase or the reversible sucrose synthase. This fructose can enter subsequent metabolism via hexokinase, or in maize kernels, by a sorbitol dehydrogenase that reversibly converts fructose + NADH to sorbitol + NAD. High levels of SDH activity suggest that kernels synthesize considerable amounts of sorbitol, but the molecular mechanism and functional role for this process have remained equivocal. To gain insights on the role of sorbitol synthesis in maize endosperm we cloned and characterized the transcriptional control of the maize sorbitol dehydrogenase (Sdh1) gene. Data indicated that Sdh1 was essentially kernel- and endosperm-specific, with maximal expression at both the mRNA and enzyme activity levels during early kernel development. Expression was elevated in high-sugar mutants (sugary1, shrunken2), also by sugar injections, and was more pronounced when transfected tissues were incubated at low oxygen concentrations. Control of Sdh1 expression in our transient assays was largely dependent on the first intron of Sdh1. We speculate that SDH activity may represent an adaptation to the high-sugar/low-oxygen environment of the endosperm. Under these conditions, the NADH-dependent reduction of fructose to sorbitol would regenerate NAD[+], thus contributing to the maintenance of the redox and energy status of the cell.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , L-Iditol 2-Desidrogenase/metabolismo , Sorbitol/farmacologia , Sacarose/farmacologia , Zea mays/efeitos dos fármacos , Zea mays/enzimologia , Genoma de Planta/genética , L-Iditol 2-Desidrogenase/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Aldose reductase catalyzes the NADPH-linked reduction of hexoses to their respective sugar-alcohols, which are involved in the pathogenesis of "sugar-cataracts". In the lenses, the reaction catalyzed by G-6-PD is the source of NADPH supply blocking sugar-alcohol formation and consequently prevents or delays the onset of "sugar-cataracts". We have investigated the effect of G-6-PD deficiency, either experimentally induced or genetically transmitted, on the sorbitol accumulation in whole cells incubated in high glucose media and on the "sugar-cataracts" formation in a galactosemic rat model. We also screened 31 Negro male adults with diabetes mellitus for red cell G-6-PD deficiency. G-6-PD deficiency produced a significant inhibition on sorbitol accumulation in rat lenses and human red cells incubated in 50 mM glucose. In the galactosemic rat model G-6-PD deficiency experimentally induced with acetaminophen delayed the development of cataracts. Finally, two diabetic individuals were G-6-PD deficient and did not show cataracts whereas cataracts were identified in six other diabetic patients.