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l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

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In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

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In order to analyze the synthesis of antioxidant and heavy metal-chelating compounds in response to copper stress, the marine alga Ulva compressa (Chlorophyta) was exposed to 10 µM copper for 7 days and treated with inhibitors of ASC synthesis, lycorine, and GSH synthesis, buthionine sulfoximine (BSO). The levels of ascorbate, in its reduced (ASC) and oxidized (DHA) forms, glutathione, in its reduced (GSH) and oxidized (GSSG) forms, and phytochelatins (PCs) were determined as well as activities of enzymes involved in ASC synthesis, L-galactose dehydrogenase (GDH) and L-galactono 1,4 lactone dehydrogenase (GLDH), and in GSH synthesis, γ-glutamylcysteine synthase (γ-GCS) and glutathione synthase (GS). The level of ASC rapidly decreased to reach a minimum at day 1 that remained low until day 7, DHA decreased until day 1 but slowly increased up to day 7 and its accumulation was inhibited by lycorine. In addition, GSH level increased to reach a maximal level at day 5 and GSSG increased up to day 7 and their accumulation was inhibited by BSO. Activities of GDH and GLDH increased until day 7 and GLDH was inhibited by lycorine. Moreover, activities of γ-GCS and GS increased until day 7 and γ-GCS was inhibited by BSO. Furthermore, PC2, PC3 and PC4, increased until day 7 and their accumulation was inhibited by BSO. Thus, copper induced the synthesis of ascorbate, glutathione and PCs in U. compressa suggesting that these compounds are involved in copper tolerance. Interestingly, U. compressa is, until now, the only ulvophyte showing ASC, GSH and PCs synthesis in response to copper excess.

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Background: Acinetobacter baumannii is an important nosocomial pathogen whose virulence factors have not been fully elucidated. Aim: To study the adherence and hemagglutinating capacity of several biotypes of Acinetobacter baumannii. Material and methods: Thirty nine strains of Acinetobacter baumannii isolated from hospitalized patients were studied. The adherence of these strains to small pieces of rat tracheal tissue was studied. Additionally, their ability to hemagglutinate human erythrocytes and the effect of D-mannose and D-galactose on the adherence and hemagglutinating capacity was assessed. Transmission electron microscopy of strains was performed looking for the presence of fimbriae. Results: All strains exhibited adherence to tissues. All strains had also D-mannose and D-galactose resistant hemagglutinating ability. Fimbriae were found in Acinetobacter baumannii and E coil cells. Conclusions: Adherence of Acinetobacter baumannii to rat tracheal tissue, apparently not related to the presence of fimbriae, may be a virulence mechanism of this bacterium

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Haptoglobina sérica em 100 doadores de sangue foi determinada utilizando-se da capacidade de ligaçäo da haptoglobina à hemoglobina; os valores médios encontrados foram de 211 ñ 82mg/dl; 38 pacientes portadores de valvopatia valvar foram também estudados e apresentaram valores significativamente mais altos (estenose: 310 ñ 128 e insuficiência 409 ñ 137mg/dl) que a populaçäo normal. A atividade da desidrogenase lática também estava aumentada em pacientes com cardiopatia valvar, indicando a presença de doença sistêmica ou eventual hemólise. Estes dados sugerem ter sido a desidrogenase lática mais sensível que a determinaçäo das haptoglobinas em detectar hemólise intravascular leve, nos doentes estudados

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