'Beta'-D-frutofuranosidases de Fusarium graminearum: produção, purificação, imobilização e determinação das propriedades bioquímicas de enzimas solúveis e secas em Spray dryer

Pós-graduação em Biotecnologia - IQ

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Theoretical studies on the conformation of beta-D-N-acetyl neuraminic acid (sialic acid)

The possible conformations of sialic acid were analysed using semi-empirical potential functions. The solid state conformation has approx. 0.2 kcal/mol higher energy than the minimum energy conformation. These studies suggest that in solution sialic acid may exist preponderantly in two different conformations which differ in the orientation of the terminal hydroxymethyl group of glycerol side-chain. The present model is consistent with 1H- and 13C-NMR data, but differs from the earlier models.

The modes of binding methyl-alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A--a theoretical approach

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Role of hydroxyl group on the mesomorphism of alkyl glycosides:synthesis and thermal behavior of alkyl 6-deoxy-beta-D-glucopyranosides

A homologous series of alkyl 6-deoxy-beta-D-glucopyranoside amphiphiles was prepared,in an effort to identify the role of hydroxyl group in the mesomorphic behavior of alkyl glycosides. Synthesis was performed by a chlorination of the sugar moiety in alkyl-beta-D-glucopyranosides with methylsulfonyl chloride in DMF, followed by a metal mediated dehalogenation to secure alkyl 6-deoxy-beta-D-glucopyranosides, wherein the alkyl chain length varied from C-9 to C-16. The mesomorphic behavior of these 6-deoxy alkyl glycosides was assessed using polarizing optical microscopy, differential scanning calorimetry and X-ray diffraction method. Whereas the lower homologues exhibited a monotropic SmA phase till sub-ambient temperatures, the higher homologues formed a plastic phase. A partial interdigitized bilaye structure of SmA phase is inferred from experimental d-spacing and computationally derived lengths of the molecules. The results were compared with those of normal alkyl glucopyranosides, retained with hydroxyl groups at C-2-C-6 carbons, and alkyl 2-deoxy-glucopyranosides, devoid of a hydroxyl group at C-2 and the comparison showed important differences in the mesomorphic behavior.(C)2010 Elsevier Ireland Ltd. All rights reserved.

Solid state structure of p-bromo phenyl 4,5,7-tri-O-benzyl-beta-D-glycero-D-talo-septanoside and an analysis of non-covalent interactions

The solid state structure of a new seven-membered sugar oxepane derivative, namely, p-bromo phenyl 4,5,7-tri-O-benzyl-beta-D-glycero-D-talo-septanoside is discussed, as determined through single crystal X-ray structural determination and in relation to their conformational features. The molecule adopts twist-chair as the preferred conformation, with conformational descriptor (TC2,3)-T-0,1. The solid state packing of molecules is governed by a rich network of non-covalent bonding originating from O-H center dot center dot center dot O, C-H center dot center dot center dot pi, C-H center dot center dot center dot Br and aromatic pi center dot center dot center dot pi interactions that stabilize the packing of molecules in the crystal. (C) 2015 Elsevier Ltd. All rights reserved.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Structural and kinetic characterization of the LPS biosynthetic enzyme D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) from Escherichia coli

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

Structural-Functional Studies of Burkholderia cenocepacia D-Glycero-beta-D-manno-heptose 7-Phosphate Kinase (HldA) and Characterization of Inhibitors with Antibiotic Adjuvant and Antivirulence Properties

As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-beta-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Purification and structural characterisation of (1 -> 3 ; 1 -> 6)-beta-D-glucans (botryosphaerans) from Botryosphaeria rhodina grown on sucrose and fructose as carbon sources: a comparative study

Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus Botryosphaeria rhodina, when grown on sucrose and fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted to trypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysis and Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl beta(1 -> 3) linkages substituted at O-6. The products obtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked beta(1 -> 6) residues. C-13-NMR spectroscopy studies showed that all glucosidic linkages were of the beta-configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%). (c) 2005 Published by Elsevier Ltd.

Structural characterization of Botryosphaeran: a (1 -> 3;1 -> 6)-beta-D-glucan produced by the ascomyceteous fungus, Botryosphaeria sp.

The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomycetcous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and C-13 NMR spectroscopy showed that all the glucosidic linkages were in the beta-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1 --> 3)-beta-(D)-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1 --> 6)-beta-linked glucosyl, and (1 --> 6)-beta-linked gentiobiosyl residues.[GRAPHICS](C) 2003 Elsevier Ltd. All rights reserved.