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.

Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase.

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.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

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.

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.

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).

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli.

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1% xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an Mr of 22 000.

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.

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.

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.

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.

Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalyzed by Bacillus (1-->3),(1-->4)-Beta-D-glucan 4-glucanohydrolase.

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.

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.

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.

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.

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.

Solvent-dependent conformational behaviour of lipochitoligosaccharides related to Nod factors.

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.

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)

Thiooligosaccharides as tools for structural biology.

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.

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.