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PubMed1977

Synthesis of p-nitrophenyl 6-O-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-beta-D-galactopyranoside and p-nitrophenyl O-beta-D-galactopyranosyl-(1 linked to 3)-O-(2-acetamido-2-dexoy-beta-D-glucopyranosyl)-(1 linked to 6)-beta-D-galactopyranoside.

Condensation of 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-alpha-D-glucopyrano)-[2',1':4,5]-2-oxazoline with p-nitrophenyl 2,3-di-O-acetyl-beta-D-galactopyranoside (4), followed by saponification of the resulting disaccharide derivative, produced p-nitrophenyl 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-D-galactopyranoside as a crystalline compound. Reaction of 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyrano]-[2',1':4,5]-2-oxazoline with 4 in a similar reaction-sequence provided the title trisaccharide compound.

Disaccharides↗
PubMed1992

Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.

Carbohydrate Sequence↗
PubMed1997

Enzymatic synthesis of the glycosides of calystegines B1 and B2 and their glycosidase inhibitory activities.

Several glycosides of calystegines B1 and B2 were synthesized by use of rice alpha-glucosidase and the whole cells of Rhodotorula lactosa, and their glycosidase inhibitory activities were investigated. Incubation of mixture of calystegine B1 and maltose with rice alpha-glucosidase gave 3-O-alpha-D-glucopyranosylcalystegine B1 (2, 11.3%). An enzymatic beta-transglucosylation reaction of calystegines B1 or B2 with cellobiose using the whole cells of R. lactosa gave 3-O-beta-D-glucopyranosylcalystegine B1 (1) (0.9%) or 4-O-beta-D-glucopyranosylcalystegine B2 (3, 11.2%), respectively, while similar beta-transgalactosylation of calystegine B2 from lactose gave 4-O-beta-D-galactopyranosylcalystegine B2 (4, 10.1%). The glycosylation of calystegines B1 and B2 markedly decreased or abolished their inhibition against beta-glucosidase, alpha- or beta-galactosidase. Compound 4 however retained more or less the potency of calystegine B2 against trehalase. Interestingly, compound 1 was a noncompetitive inhibitor of rice alpha-glucosidase, with a Ki value of 0.9 +/- 0.1 microM.

Animals↗
PubMed1997

Diastereoselective resolution of 6-substituted glycosides via enzymatic hydrolysis.

The diastereoselectivity of the enzymatic hydrolyses of 4-nitrophenyl 6-deoxy-6-methyl-(R)- and (S)-sulfinyl-beta-D-galactopyranoside (1a,b), 4-nitrophenyl 7-deoxy-D- and L-glycero-beta-D-galacto-heptopyranoside (2a,b) and 4-nitrophenyl 6,7-anhydro-D- and L-glycero-beta-D-galacto-heptopyranoside (3a,b) was investigated using a range of crude glycosidase preparations. It was shown that the enzymes display a high degree of discrimination between diastereomers thereby demonstrating the utility of glycosidases for the diastereomeric resolution of unnatural 6-substituted monosaccharide derivatives.

Chromatography, High Pressure Liquid↗
PubMed1998

Synthesis of aryl 3'-sulfo-beta-lactosides as fluorogenic and chromogenic substrates for ceramide glycanases.

We have previously reported that 4-methylumbelliferyl 6'-O-benzyl-beta-lactoside (2) is a useful substrate for a fluorometric assay of ceramide glycanase (CGase) (L.-X. Wang, N. V. Pavlova, S.-C. Li, Y.-T. Li and Y. C. Lee, Glycoconjugate J., 13 (1996) 359-365). The introduction of a 6-O-benzyl group at the terminal Gal efficiently protected the substrate from its hydrolysis by exo-galactosidase, permitting the assay of CGase in crude biological materials. However, a drawback of this substrate is its low water-solubility and relatively high Km (at a mM level). Introduction of a sulfate group into 4-methylumbelliferyl beta-lactoside (1) led to the formation of 4-methylumbelliferyl 3'-O-sulfo-beta-lactoside (3), which was found to be a more effective substrate than 2. Moreover, the presence of a 3'-O-sulfate group not only increases the water solubility tremendously, but also protects the substrate from cleavage by exo-beta-galactosidase as the 6'-O-benzyl group in 2 does. In addition to the fluorogenic substrate (3), two sulfated chromogenic substrates, N-tetradecanoyl-4-O(3'-sulfo-beta-lactosyl)-3-nitro-L-tyrosine methyl ester (9) and 2-N-(tetradecanoylamino)-4-nitro-phenyl 3'-sulfo-beta-lactoside (12), were synthesized and their suitability for a photometric assay of CGase was evaluated. Substrates 9 and 12, with a long fatty acid chain attached to the aglycon part, have a Km value close to that of the natural substrate GM1 (at a microM level).

Chromogenic Compounds↗
PubMed2000

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase.

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.

Amino Acid Sequence↗
PubMed2002

Maltooligosaccharide disproportionation reaction: an intrinsic property of amylosucrase from Neisseria polysaccharea.

Amylosucrase from Neisseria polysaccharea (AS) is a remarkable transglycosidase of family 13 of the glycoside hydrolases that catalyses the synthesis of an amylose-like polymer from sucrose and is always described as a sucrose-specific enzyme. Here, we demonstrate for the first time the ability of pure AS to catalyse the disproportionation of maltooligosaccharides by cleaving the alpha-1,4 linkage at the non-reducing end of a maltooligosaccharide donor and transferring the glucosyl unit to the non-reducing end of another maltooligosaccharide acceptor. Surprisingly, maltose, maltotriose and maltotetraose are very poor glucosyl donors whereas longer maltooligosaccharides are even more efficient glucosyl donors than sucrose. At least five glucose units are required for efficient transglucosylation, suggesting the existence of strong binding subsites, far from the sucrose binding site, at position +4 and above.

Carbohydrate Conformation↗
PubMed2002

Biochemical characterization of Thermotoga maritima endoglucanase Cel74 with and without a carbohydrate binding module (CBM).

The genome of the hyperthermophilic bacterium Thermotoga maritima (Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino-terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide in Escherichia coli and found to be optimally active at pH 6, 90 degrees C, with a melting temperature of approximately 105 degrees C. The cel74 gene was previously found to be induced during T. maritima growth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile, Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan-based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.

Amino Acid Sequence↗
PubMed2003

Molecular identification of wheat endoxylanase inhibitor TAXI-I1, member of a new class of plant proteins.

Triticum aestivum endoxylanase inhibitors (TAXIs) are wheat proteins that inhibit family 11 endoxylanases commonly used in different (bio)technological processes. Here, we report on the identification of the TAXI-I gene which encodes a mature protein of 381 amino acids with a calculated molecular mass of 38.8 kDa. When expressed in Escherichia coli, the recombinant protein had the specificity and inhibitory activity of natural TAXI-I, providing conclusive evidence that the isolated gene encodes an endoxylanase inhibitor. Bioinformatical analysis indicated that no conserved domains nor motifs common to other known proteins are present. Sequence analysis revealed similarity with a glycoprotein of carrot and with gene families in Arabidopsis thaliana and rice, all with unknown functions. Our data indicate that TAXI-I belongs to a newly identified class of plant proteins for which a molecular function as glycoside hydrolase inhibitor can now be suggested.

Amino Acid Sequence↗
PubMed2004

Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increased activity.

Amylosucrase is a glucosyltransferase belonging to family 13 of glycoside hydrolases and catalyses the formation of an amylose-type polymer from sucrose. Its potential use as an industrial tool for the synthesis or the modification of polysaccharides, however, is limited by its low catalytic efficiency on sucrose alone, its low stability, and its side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling, and selective screening (directed evolution) was started, in order to generate more efficient variants of the enzyme. A convenient zero background expression cloning strategy was developed. Mutant gene libraries were generated by error-prone polymerase chain reaction (PCR), using Taq polymerase with unbalanced dNTPs or Mutazyme trade mark, followed by recombination of the PCR products by DNA shuffling. A selection method was developed to allow only the growth of amylosucrase active clones on solid mineral medium containing sucrose as the sole carbon source. Automated protocols were designed to screen amylosucrase activity from mini-cultures using dinitrosalicylic acid staining of reducing sugars and iodine staining of amylose-like polymer. A pilot experiment using the described mutagenesis, selection, and screening methods yielded two variants with significantly increased activity (five-fold under the screening conditions). Sequence analysis of these variants revealed mutations in amino acid residues which would not be considered for rational design of improved amylosucrase variants. A method for the characterisation of amylosucrase action on sucrose, consisting of accurate measurement of glucose and fructose concentrations, was introduced. This allows discrimination between hydrolysis and transglucosylation, enabling a more detailed comparison between wild-type and mutant enzymes.

Catalysis↗
PubMed2004

Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121.

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935-941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5-4x10(5) times reduction of total sucrase activity.

Amino Acid Motifs↗
PubMed2004

Essential role of the family-22 carbohydrate-binding modules for beta-1,3-1,4-glucanase activity of Clostridium stercorarium Xyn10B.

Clostridium stercorarium Xyn10B is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules consecutively from the N-terminus. To investigate the role of the family-22 CBMs, truncated proteins were constructed: a recombinant catalytic module polypeptide (rCD), a CBM polypeptide composed of two family-22 CBMs (rCBM) and a polypeptide composed of the family-22 CBMs and the catalytic module (rCBM-CD). We found that rCBM-CD was highly active toward beta-1,3-1,4-glucan; however, rCD was negligibly active toward the same substrate. The V(max)/K(m) value of rCBM-CD for beta-1,3-1,4-glucan was 7.8 times larger than that for oat-spelt xylan, indicating that rCBM-CD should be specified as a beta-1,3-1,4-glucanase rather than a xylanase despite the fact that family-10 catalytic modules are well-known xylanase modules. These results indicate that the family-22 CBMs in rCBM-CD are essential for hydrolysis of beta-1,3-1,4-glucan.

Clostridium↗
PubMed1998

A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants.

A scheme is proposed for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. These enzymes are predominantly beta-1,4-glycanases. The scheme is based on the classification of the catalytic domains of glycoside hydrolases into families of related amino acid sequences. The new designation for an enzyme indicates its family and, because all members of a family have these characteristics in common, its three-dimensional fold and stereospecificity of hydrolysis. The scheme is intended to simplify comparison of the systems of enzymes produced by different microorganisms for the hydrolysis of plant cell walls.

Binding Sites↗