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BACKGROUND: Barley beta-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta-D-glucans and beta-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. RESULTS: When conduritol B epoxide and 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Odelta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I), 4(III), 4(V)-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the -1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 A) with the Oepsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C(1) conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. CONCLUSIONS: X-ray crystallography is used to define the three key intermediates during catalysis by beta-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex.

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Many three-dimensional structures of retaining beta-D-glycoside hydrolases have been determined, yet oligosaccharide complexes in which the ligand spans the catalytic centre are rare. Those that have been reported so far have revealed two modes of binding: those in which the substrate adopts a distorted skew-boat or envelope conformation in the -1 subsite, reflecting the distortion observed during the catalytic cycle, and those which bypass the true catalytic centre and thus lie in a non-productive manner across the -1 subsite. The three-dimensional structure of a retaining endocellulase, Bacillus agaradhaerens Cel5A, in complex with methyl 4,4(II),4(III),4(IV)-tetrathio-alpha-cellopentoside falls into this latter category. The 1.1 A structure reveals the binding of five pyranosides, all in the (4)C(1) chair conformation, occupying the -3, -2, +1 and +2 subsites whilst evading the catalytic machinery located in the true -1 subsite. Such binding is in marked contrast to the structure of another retaining endocellulase, the Fusarium oxysporum Cel7B, the identical ligand in which displayed a distorted skew-boat conformation at the active centre. These two binding modes may reflect different steps in the binding and catalytic process.

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Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

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A new class of inhibitors for beta-D-glycoside hydrolases, in which a single alpha-(1-->4)-glycosidic bond is incorporated into an otherwise all-beta-(1-->4)-linked oligosaccharide, is described. Such mixed beta/alpha-linkage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning either side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed alpha/beta-D-tetrasaccharide acts competitively on a number of cellulases, displaying inhibition constants in the range of 40-300 microM. Using the Bacillus agaradhaerens enzyme Cel5A as a model system, one such mixed beta/alpha-cellooligosaccharide, methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside, displays a K(i) value of 100 microM, an inhibition at least 150 times better than is observed with an equivalent all-beta-linked compound. The three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4(II),4(III)-dithio-alpha-cellobiosyl-(1-->4)-beta-cellobioside has been determined at 1.8 A resolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the (4)C(1) chair conformation, occupies the -3, -2 and +1 subsites whilst evading the catalytic (-1) subsite. Such "by-pass" compounds offer great scope for the development of a new class of beta-D-glycoside hydrolase inhibitors.

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Oligosaccharides in which at least one glycosidic oxygen atom is replaced with a sulfur atom can be routinely synthesized and act as competitive inhibitors of various glycoside hydrolases. Recent studies using both X-ray crystallography and other biophysical techniques provide structural insight into binding, recognition, and the catalytic mechanism of action of these enzymes.

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Glycosynthases are engineered glycosidases which are hydrolytically inactive yet efficiently catalyse transglycosylation reactions of glycosyl fluoride donors, and are thus promising tools for the enzymatic synthesis of oligosaccharides. Two endo-glycosynthases, the E134A mutant of 1,3/1,4-beta-glucanase from Bacillus licheniformis and the E197A mutant of cellulase Cel7B from Humicola insolens, were used in coupled reactions for the stepwise synthesis of hexasaccharide substrates of 1,3/1,4-beta-glucanases. Because the two endo-glycosynthases show different specificity, towards laminaribiosyl and cellobiosyl donors, respectively, the target hexasaccharides were prepared by condensation of the corresponding disaccharide building blocks through sequential addition of the glycosynthases in a "one-pot" process. Different strategies were used to achieve the desired transglycosylation between donor and acceptor in each step, and to prevent unwanted elongation of the first condensation product and polymerization (self-condensation) of the donor: 1) selection of disaccharide donors differing in the configuration of the hydroxyl substituent normally acting as acceptor, 2) temporary protection of the polymerizable hydroxyl group of the donor, or 3) addition of an excess of acceptor to decrease the probability that the donor can act as an acceptor. The best procedure involved the condensation of alpha-lactosyl or 4II-O-tetrahydropyranyl-alpha-cellobiosyl fluorides with alpha-laminaribiosyl fluoride, catalyzed by E197A Cel7B, to give tetrasaccharide fluorides, which were then the donors for in situ condensation with methyl beta-cellobioside catalyzed by E134A 1,3/1,4-beta-glucanase. After isolation, the final hexasaccharides Gal/beta4Glcbeta4Glcbeta3Glcbeta4Glcbeta4Glcbeta-OMe and Glcbeta4Glcbeta4Glcbeta3Glcbeta4Glcbeta4-Glcbeta-OMe were obtained in 70-80% overall yields.

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Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase. The active site is composed of a 25 A long tunnel which is followed by an open cleft. During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage. To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose. Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed. The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit. These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel. Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking. It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

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A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII, NIV-tetra-acetyl-chitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphtalene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo-chitinase and chitobiosidase activities could be specifically assayed by monitoring the variation of fluorescence after enzymatic hydrolysis of this substrate. Chitobiases and N-acetyl-beta-glucosaminidases are not active on the compound, the presence of a bulky chromogenic group at the 2 position of the nonreducing end of the subtrate preventing the binding and thus hydrolysis by these two exo-enzymes. The observation that chitobiosidases are able to hydrolyse a chitooligosaccharide functionalized on both extremities demonstrates the possibility of an endo-action for this class of chitinases, which are generally classified as exo-enzymes. This fluorogenic chitooligosaccharide should prove to be very useful for the detection and the convenient assay of chitinolytic activities at nanomolar concentrations.

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The solution conformation of two lipooligosaccharides related to Nod factors or lipochitoligosaccharides have been analysed by 1D and 2D 1H and 13C NMR spectroscopy, molecular mechanics and dynamics calculations. The obtained data indicate that the glycosidic torsion angles have restricted fluctuations, but may adopt a variety of shapes. Remarkably, the relative orientation of the fatty acid chain towards the oligosaccharide backbone is solvent dependent. In water solution, the acyl residue and the oligosaccharide adopt a quasi-parallel orientation for a significant amount of time.

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BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

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Phosphorylases are key enzymes of carbohydrate metabolism. Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered. How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase. The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-alpha-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 A resolution; and the 2.1 A resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P). The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites -1 to +4. Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate. The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis. In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and +1, the site of catalysis, from the preferred conformation for alpha(1-4)-linked glucosyl polymers.

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Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.

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Enzymatic hydrolysis of barley (1-->3),(1-->4)-beta-D-glucan using a recombinant (1-->3),(1-->4)-beta-glucanase from Bacillus licheniformis gives Glc beta 4Glc beta 3Glc isolated after acetylation in 49% yield. Conventional treatment produced the corresponding beta-fluoride which was carefully de-O-acetylated. A transglycosylation reaction with this substrate, catalyzed by the title enzyme, gave Glc beta 4Glc beta 3Glc beta 4Glc beta 4Glc beta 3Glc in 20% yield.

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The catalytic domain of the CeIF processive endocellulase, a family 48 glycosyl hydrolase from Clostridium cellulolyticum has been crystallized in the presence of a newly synthesized inhibitor (methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside), by vapour diffusion, using PEG as a precipitant. The protein crystallizes in the orthorhombic P212121 space group and diffracts to a resolution of 2.0 A. The unit-cell parameters are a = 61.4, b = 84.5, c = 121.9 A.

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The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose. One of the three major components of the cellulosome is the processive endocellulase CelF. The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 A resolution. This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases. The structure consists of an (alpha alpha)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel. One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft. The active centre is located in a depression at the junction of the cleft and tunnel regions. Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230. The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain.

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The binding to glucoamylase 1 from Aspergillus niger (GA1) of a series of four synthetic heterobidentate ligands of acarbose and beta-cyclodextrin (beta-CD) linked together has been studied by isothermal titration calorimetry. GA1 consists of a catalytic and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region. Acarbose is a strong inhibitor of glucoamylase and binds exclusively in the catalytic site, while the cyclic starch mimic beta-CD binds exclusively to the two sites of SBD. No spacer or spacer arms of 14, 36, and 73 A in their extended conformations connect acarbose and beta-CD. These compounds were used as probes for bidentate ligand binding to both domains in order to estimate the distance between the catalytic site and the SBD binding site in solution. DeltaH of binding of the four heterobidentate ligands is within experimental uncertainty equal to the sum of DeltaH of binding of free acarbose and beta-CD, indicating ligand binding to both domains. However, the binding constants are 4-5 orders of magnitude smaller than for the binding of acarbose (K approximately 10(12) M-1), increasing with spacer length from 2 x 10(7) M-1 for no spacer to 1 x 10(8) M-1 for the 73 A spacer. Subsequent titrations with beta-CD of the glucoamylase-bidentate ligand complexes revealed that only one of the two binding sites of SBD was vacant. Further titrations with acarbose to these mixtures showed complete displacement of the acarbose moiety of the bidentate ligands from the catalytic sites. These experiments show that the bidentate ligands bind to both the catalytic domain and SBD. The weakening of the bidentate ligand binding compared to acarbose is a purely entropic effect point to steric hindrance between SBD and the beta-CD moiety. To test this, titrations of glucoamylase 2, a form containing the catalytic domain and the linker region but lacking SBD, with the bidentate ligands were carried out. The results were indistinguishable from the binding of free acarbose. Thus, the reduced affinity of the bidentate ligands observed with GA1 stems from interactions due to SBD. The results show that the catalytic and starch-binding sites are in close proximity in solution and thus indicate considerable flexibility of the linker region.

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Crystals of the inactive mutant Glu257-->Ala of cyclodextrin glycosyltransferase were soaked with the cyclodextrin (CD) derivative S-(alpha-D-glucopyranosyl)-6-thio-beta-CD. The structural analysis showed its beta-CD moiety with no density indication for the exocyclic glucosyl unit. For steric reasons, however, the position of this unit is restricted to be at only two of the seven glucosyl groups of beta-CD. The analysis indicated that the enzyme can cyclize branched alpha-glucans. The ligated beta-CD moiety revealed how the enzyme binds its predominant cyclic product. The conformation of the ligated beta-CD was intermediate between the more symmetrical conformation in beta-CD dodecahydrate crystals and the conformation of a bound linear alpha-glucan chain. Its scissile bond was displaced by 2.8 A from the position in linear alpha-glucans. Accordingly, the complex represents the situation after the cyclization reaction but before diffusion into the solvent, where a more symmetrical conformation is assumed, or the equivalent state in the reverse reaction. Furthermore, a unifying nomenclature for oligosaccharide-binding subsites in proteins is proposed.

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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.

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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.

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