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H Driguez

Publications and source records attributed to H Driguez.

At least 37 records · Page 2Linked to original sources
PubMed1998

Xyloglucan octasaccharide XXLGol derived from the seeds of hymenaea courbaril acts as a signaling molecule

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-beta-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2, 4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an alpha-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced alpha-l-fucosidase.

Journal Article↗
PubMed1997

Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli.

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

Acetylation↗
PubMed1997

The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase.

BACKGROUND: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-beta-D-glucosides called glucosinolates. Myrosinase and glucosinolates, which are stored in different tissues of the plant, are mixed during mastication generating toxic by-products that are believed to play a role in the plant defence system. Whilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. This intriguing enzyme, which shows sequence similarities with O-glycosidases, offers the opportunity to analyze the similarities and differences between enzymes hydrolyzing S- and O-glycosidic bonds. RESULTS: The structures of native myrosinase from white mustard seed (Sinapis alba) and of a stable glycosyl-enzyme intermediate have been solved at 1.6 A resolution. The protein folds into a (beta/alpha)8-barrel structure, very similar to that of the cyanogenic beta-glucosidase from white clover. The enzyme forms a dimer stabilized by a Zn2+ ion and is heavily glycosylated. At one glycosylation site the complete structure of a plant-specific heptasaccharide is observed. The myrosinase structure reveals a hydrophobic pocket, ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic nucleophile of myrosinase. CONCLUSIONS: The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.

Amino Acid Sequence↗
PubMed1997

A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases.

Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. The substrate, which carries a 5-(2-aminoethylamino)-1-naphthalenesulfonate group on the non-reducing end and an indolethyl group on the reducing end, was prepared from beta-lactosyl fluoride and indolethyl beta-cellobioside by a chemoenzymatic approach using the transglycosylating activity of endoglucanase I of Humicola insolens as the key step. The bifunctionalized substrate has been used for the determination of the catalytic constants of H. insolens endoglucanase I and cellobiohydrolases I and II; this substrate could be of general use to measure the kinetic constants of cellulases able to act on oligomers of degree of polymerization <5. The data also provide evidence that cellobiohydrolases I and II are able to degrade an oligosaccharide substrate carrying non-carbohydrate substituents at both ends.

Carbohydrate Conformation↗
PubMed1997

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution.

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).

Acarbose↗
PubMed1997

Transfer reactions catalyzed by cyclodextrin glucosyltransferase using 4-thiomaltosyl and C-maltosyl fluorides as artificial donors.

Cyclodextrin glycosyltransferase enzyme from Bacillus circulans catalyzed the effective conversion of 4-thio-alpha-maltosyl fluoride into cyclo-alpha-(1-->4(2))-thiomalto -tetraoside, -pentaoside, -hexaoside and linear hemithiomaltooligosaccharides. However, under the same conditions, C-maltosyl fluoride afforded only linear modified maltotetraose, maltohexaose and maltooctaose in moderate yield.

Bacillus↗
PubMed1996

Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: substrate distortion gives rise to the preferred axial orientation for the leaving group.

Endoglucanase I (EG I) is a cellulase, from glycosyl hydrolase family 7, which cleaves the beta-1,4 linkages of cellulose with overall retention of configuration. The structure of the EG I from Fusarium oxysproum, complexed to a nonhydrolyzable thiooligosaccharide substrate analogue, has been determined by X-ray crystallography at a resolution of 2.7 A utilizing the 4-fold noncrystallographic symmetry present in the asymmetric unit. The electron density map clearly reveals the presence of three glucosyl units of the inhibitor, consistent with the known number of sugar-binding subsites, located at the active site of the enzyme in the -2, -1, and +1 subsites, i.e., actually spanning the point of enzymatic cleavage. The pyranose ring at the point of potential enzymatic cleavage is clearly distorted from the standard 4C1 chair as was originally suggested for beta-retaining enzymes by Phillips [Ford, L.O., Johnson, L.N., Machin, P. A., Phillips, D.C., & Tijan, T. (1974) J. Mol. Biol, 88, 349-371]. The distortion observed goes beyond the "sofa" conformation observed in previous studies and results in a conformation whose salient feature is the resulting quasi-axial orientation for the glycosidic bond and leaving group, as predicted by stereoelectronic theory. An almost identical conformation has recently been observed in a complex of chitobiase with its unhydrolyzed substrate [Tews, I., Perrakis, A., Oppenheim, A., Dauter, Z., Wilson, K. S., & Vorgias, C. E. (1996) Nat. Struct. Biol. 3, 638-648]. The striking similarity between these two complexes extends beyond the almost identical pyranose ring distortion. The overlap of the two respective sugars places the enzymatic nucleophile of endoglucanase I in coincidence with the C2 acetamido oxygen of N-acetylglucosamine in the catalytic site of the chitobiase, substantiating the involvement of this group in the catalytic mechanism of chitobiase and related chitinolytic enzymes. The endoglucanase I complex with the thiosaccharide substrate analogue clearly illustrates the potential of nonhydrolyzable sulfur-linked oligosaccharides in the elucidation of substrate binding and catalysis by glycosyl hydrolases.

Cellulase↗
PubMed1996

Mechanism-based inhibition and stereochemistry of glucosinolate hydrolysis by myrosinase.

Myrosinase is a particular glucosidase which hydrolyzes a variety of plant 1-thio-beta-D-glucosides known as the glucosinolates. This enzyme, which is the only glycosidase able to hydrolyze these naturally occurring thioglucosides, has been found previously to display strong sequence similarities with family 1 O-glycosidases. Myrosinase therefore offers the opportunity to compare the mechanism of enzymatic cleavage of S- vs O-glycosidic bonds. The stereochemistry of hydrolysis of sinigrin by Sinapis alba myrosinase was followed by 1H NMR and the enzyme was found to operate with a mechanism retaining the anomeric configuration at the cleavage point exactly like the related O-glycosidases found in family 1. Myrosinase was readily inactivated by 2-deoxy-2-fluoroglucotropaeolin with inactivation kinetic parameters of Ki = 0.9 mM and ki = 0.083 min-1. Reactivation kinetic parameters were determined in buffer only, with k(react) = 0.015 h-1 and t1/2 = 46 h, and also in the presence of acceptors of transglycosylation. No significant changes were observed in the presence of methyl beta-D-glucoside, but with azide anion the half-life of reactivation was found to be reduced to t1/2 = 20 h. These results suggest that myrosinase inhibition by 2-deoxy-2-fluoroglucotropaeolin occurs via the accumulation of a long-life glucosyl-enzyme intermediate and that the catalytic machinery of the enzyme is composed of only one catalytic residue, a nucleophilic glutamate, while the acid catalyst residue found in the corresponding O-glycosidases is missing.

Glucosinolates↗
PubMed1996

Design and chemoenzymatic synthesis of thiooligosaccharide inhibitors of 1,3:1,4-beta-D-glucanases.

A successful chemoenzymatic synthesis of oligosaccharides with an interglucosidic sulfur atom as inhibitors of 1,3:1,4-D-glucanases is described. The key compound 3a was synthesized from acetylated 1-thio-beta-laminaribiose 4 and the methyl 4'-O-triflyl-lactoside 5. After de-O-acylation, the tetrasaccharide 3b was used as an acceptor and glucose-1-P as a donor in a phosphorolytic elongation catalysed by cellodextrin phosphorylase from Clostridium thermocellum. The expected pentasaccharide 2a and hexasaccharide 1 were isolated in 56% and 13% yield, respectively. As expected, the thiooligosaccharides 1, 2a, and 3b were resistant to enzymatic cleavage by 1,3:1,4-beta-D-glucanase isolated from Bacillus licheniformis. Furthermore, they have been shown to act as competitive inhibitors of the hydrolysis of the chromophoric trisaccharide substrate 11 by this enzyme.

Binding Sites↗
PubMed1995

Chemoenzymatic synthesis of 6 omega-S-alpha-D-glucopyranosyl-6 omega-thiomaltooligosaccharides: their binding to Aspergillus niger glucoamylase G1 and its starch-binding domain.

A coupling reaction of cyclodextrin glucosyltransferase (CGTase) with glucose and 6-deoxy-6-iodo-cyclomaltoheptaose (1), in the presence of glucoamylase, followed by acetylation, led to a convenient synthesis of acetylated 6III-deoxy-6III-iodo-maltotriose (2) and 6IV-deoxy-6IV-iodomaltotraose (3). Nucleophilic displacement of the iodine atom of these protected maltotriose and maltotetraose analogs by the activated form of 2,3,4,6-tetra-O-acetyl-1-S- acetyl-1-thio-alpha-D-glucose (4) afforded peracetylated 6III-S-alpha-D-glucopyranosyl-6III-thiomaltotriose (5) and 6IV-S-alpha-D-glucopyranosyl-6IV-thiomaltotetraose (6) in high yield. The interaction of OH-free tetra- and penta-saccharides (7 and 8) with both glucoamylase G1 from Aspergillus niger as well as its isolated starch-binding domain fragment were studied by UV difference spectroscopy. It was found that the starch-binding domain has higher affinity for 7 and 8 than for maltotetraose and maltopentaose.

Aspergillus niger↗
PubMed1995

Sucrose analogues modified at position 3: chemoenzymatic synthesis and inhibition studies of dextransucrases.

Conditions for the large-scale (molar) oxidation of sucrose by Agrobacterium tumefaciens were improved, thus leading to homogeneous solutions of 3-ketosucrose in 40% yield. Treatment of this solution with hydroxylamine or methoxylamine afforded the corresponding oximes 3a and 3b (isolated as acetates) in excellent yield. Dissolving-metal reduction of these oximes gave mixtures of amino disaccharides in which the gluco epimer (3-amino-3-deoxysucrose) was predominant. A more efficient approach to this amino sucrose was provided by the highly stereoselective hydrogenation of 3-ketosucrose peracetate (7), which gave exclusively the allo isomer 8 (2,4,6-tri-O-acetyl-alpha-D-allopyranosyl 1,3,4,6-tetra-O-acetyl-beta-D-fructofuranoside). Upon reaction with lithium azide, the triflate derived from 8, compound 9, afforded 3-azido-3-deoxysucrose peracetate (10) which was converted into 3-amino-3-deoxysucrose (12). The reaction of triflate 9 with potassium ethylxanthate led to a mixture of products (the expected 3-S-ethoxythiocarbonyl-3-thiosucrose derivative and the peracetates of 3-thiosucrose and of 3-thiosucrose disulfide), which could be all converted into 3-thiosucrose (17). Sucrose analogues 12 and 17 were not substrates of dextransucrases from various strains of L. mesenteroides, nor did they participate in glycosyl transfer reactions to an acceptor (maltose). Compounds 3a and 12 were found to be strong competitive inhibitors of the dextran synthesis process (dextransucrase from strain B-1397). These results indicate that 3a and 12 compete effectively with sucrose for the sucrose binding site but are unable to participate as glycosyl donors in the polymerization or glycosyl-transfer processes.

Agrobacterium tumefaciens↗
PubMed1994

Thermodynamics of ligand binding to the starch-binding domain of glucoamylase from Aspergillus niger.

The thermodynamics of ligand binding to the starch-binding domain (SBD) of glucoamylase from Aspergillus niger has been studied using titration calorimetry. The ligand binding was studied both with the SBD fragment as well as glucoamylase G1 which contains both a catalytic domain and SBD. The ligands were beta-cyclodextrin and three thiopanose analogues [panose = alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glcp] each including an alpha-(1-->6) thioglycosidic linkage at the non-reducing end. beta-Cyclodextrin binds more strongly than the thiopanose analogues and these have a slightly increasing binding constant with chain length. The reactions are enthalpy-driven with unfavourable contributions from entropy and the variations in enthalpy and entropy compensate each other linearly. SBD was shown to have two binding sites that appear to bind identically and independently in the isolated binding domain, whereas they interact with each other in a negatively cooperative fashion when the catalytic domain of glucoamylase is present (glucoamylase G1). In glucoamylase G1 one site of SBD has an increased binding constant compared to the SBD fragment, whereas the other has the same association constant. The change in binding constant and induced cooperativity were not due to interactions with the catalytic binding site, since binding of beta-cyclodextrin was the same both when the catalytic site was occupied by the strong inhibitor acarbose and when the catalytic site was free.

Amino Acid Sequence↗
PubMed1992

4-Thiocellooligosaccharides. Their synthesis and use as ligands for the separation of cellobiohydrolases of Trichoderma reesei by affinity chromatography.

4-Aminophenyl 1,4-dithio-beta-cellobioside (6) was obtained by treatment of methyl 2,3,6-tri-O-benzoyl-4-O-triflyl-alpha-D-galactopyranoside with the sodium salt of 1-thio-beta-D-glucopyranose, followed by acetolysis and glycosylation of the corresponding bromide with 4-aminobenzenethiol and subsequent deacylation. A similar synthesis starting with the 1-thiolate of 1,4-dithio-beta-cellobiose led to the trisaccharide 4-aminophenyl 1,4,4'-trithiocellotrioside (16). The 4-acetamidophenyl di- and tri-thiocellooligosaccharides were found to be excellent competitive inhibitors of the hydrolysis of 4-methylumbelliferyl beta-lactoside with respective Ki values of 25 and 6.5 mM. The two 4-aminophenyl oligosaccharides 6 and 16 were coupled to CH-Sepharose 4B, and the affinity gels were used for the purification of cellobiohydrolases from a crude commercial cellulolytic extract of T. reesei. Cellobiohydrolases I or II were selectively desorbed from gels bearing ligands 6 and 16.

Carbohydrate Sequence↗
PubMed1992

Spacer-modified disaccharide and pseudo-trisaccharide methyl glycosides that mimic maltotriose, as competitive inhibitors for pancreatic alpha-amylase: a demonstration of the "clustering effect".

The synthesis is reported of methyl 4,4'-dithio-alpha-maltotrioside (12) and the spacer-modified disaccharide glycosides methyl 4-S-(4-alpha-D-glucopyranosylthio-2-hydroxybutyl)-4-thio-alpha -D-glucopyranoside (20) and methyl 4-S-[(1,5/4,6)- and (4,6/1,5)-4-alpha-D-glucopyranosylthio-5,6-dihydroxy-2- cyclohexen-1-yl]-4-thio-alpha-D-glucopyranoside (29a/b), which are analogues of methyl alpha-maltotrioside. The Ki values for alpha-amylase for these compounds were determined as were those of methyl alpha-maltotrioside and maltose.

Carbohydrate Sequence↗
PubMed1992

Derivatives of di-O-octanoylglycerol and mono-O-octylglycerol as modulators of protein kinase C and diacylglycerol kinase activities.

Twelve analogs of 1,2-di-O-octanoylglycerol modified at C-3 and three quaternary N-alkyl-ammonium derivatives of glycerol were synthesized. The compounds were tested in vitro as potential modulators of the calcium activated, phospholipid dependent protein kinase C (PKC) and diacylglycerol (DAG) kinase activities in order to understand the molecular interactions of these enzymes with their natural activators, inhibitors, or substrates. PKC activity was assayed by measuring histone H1 phosphorylation, and the compounds synthesized were tested either in the presence (inhibitors) or in the absence (activators) of 1,2-di-O-octanoylglycerol analogs with the phosphatidylserine/Ca2+ mixture. DAG kinase activity was measured by the incorporation of phosphate into 1,2-di-O-oleoyl-sn-glycerol in the presence of the various analogs synthesized. In regard to PKC activity, the assays revealed that 1,2-di-O-octanoylglycerol analogs are inactive when modified at C-3 with groups which do not permit hydrogen bonding. Under our conditions, di-O-octanoylthioglycerol, which has been reported as inactive, was able to activate PKC in the presence of phosphatidylserine. It has been shown to give a synergistic activation with diacylglycerol and had no affinity for the phorbol ester receptor binding site, suggesting that O-octanoylthioglycerol interacts with the enzyme at a different site from the phorbol ester receptor binding site. PKC and DAG kinase activities are inhibited by N-alkyl-ammonium compounds (IC50 24 microM) only when either two 8-carbon alkyl or acyl chains are present at the 1- and 2-positions of the glycerol backbone.(ABSTRACT TRUNCATED AT 250 WORDS)

Diacylglycerol Kinase↗
PubMed1992

Photolabile derivatives of maltose and maltotriose as ligands for the affinity labelling of the maltodextrin-binding site in porcine pancreatic alpha-amylase.

The 3-azibutyl group was linked through sulfur to the anomeric position of maltose and maltotriose to yield the photolabile thioglycosides 3-azibutyl 1-thio-alpha-maltoside (11) and 3-azibutyl 1-thio-alpha-maltotrioside (12), and to the 4'- and 6'-position of maltose to give the thioethers 4'-S-(3-azibutyl)-4'-thiomaltose (8) and 6'-S-(3-azibutyl)-6'-thiomaltose (15). All four compounds were good competitive inhibitors of the action of porcine pancreatic alpha-amylase. Compound 12 irreversibly deactivated the enzyme to approximately 100% when irradiated together with the protein. The other compounds were much less effective. It is likely that separate areas of the enzyme binding site are chemically modified by the different ligands.

Affinity Labels↗
PubMed1992

Chemoenzymatic synthesis of modified maltooligosaccharides from cyclodextrin derivatives.

Methyl and p-nitrophenyl alpha-maltooligosaccharides with a 3,6-anhydro ring on the fourth glucosyl residue, starting from the reducing end, were prepared. Enzymatic coupling catalyzed by CGTase, between 3A,6A-anhydrocyclomaltohexaose and methyl or p-nitrophenyl alpha-D-glucosides led to maltohepatosides. When miglitol, a nojirimycin analogue was used, maltooligosaccharides with miglitol at the reducing end were also obtained. After glucoamylase digestion, maltopentaosides with a 3,6-anhydro glucose as antepenultimate unit were produced in good yield. The same methyl maltopentaoside was also obtained when 3A,6A-anhydrocyclomaltoheptaose was incubated with methyl alpha-D-glucoside and CGTase, glucoamylase, glucose oxidase and catalase. These results provided new information about the specificity of the subsites of CGTase.

Cyclodextrins↗