Convenient enzymatic synthesis of a p-nitrophenyl oligosaccharide series of sialyl N-acetyllactosamine, sialyl Le x and relevant compounds

From the beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (1) prepared by the transglycosylation of beta-galactosidase from Bacillus circulans, alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (9) and alpha-D-Neu5Ac-(2-->6)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (10) were effectively synthesized with an equimolar ratio of CMP-Neu5Ac by recombinant rat alpha-(2-->3)-N-sialyltransferase and rat liver alpha-(2-->6)-N-sialyltransferase, respectively. The former enzyme also transferred effectively the Neu5Ac residue from CMP-Neu5Ac to the location of OH-3 in the non-reducing terminal of beta-D-Gal-(1-->4)-beta-D-Gal-OC6H4NO2-p or beta-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p, while the latter enzyme did not. In the case of equimolar ratio of GDP-Fuc/acceptor, 1 and 9 were further fucosylated quantitatively to form beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (14) and alpha-D-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-(alpha-l-Fuc-(1-->3)-)-GlcNAc-OC6H4NO2-p (13) by recombinant human alpha-(1-->3)-fucosyltransferase VII, respectively.     

Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.     

Enzymatic synthesis of alpha-L-fucosyl-N-acetyllactosamines and 3'-O-alpha-L-fucosyllactose utilizing alpha-L-fucosidases

An alpha-L-fucosidase from porcine liver produced alpha-L-Fuc-(1-->2)-beta-D-Gal-(1-->4)-D-GlcNAc (2'-O-alpha-L-fucosyl-N-acetyllactosamine, 1) together with its isomers alpha-L-Fuc-(1-->3)-beta-D-Gal-(1-->4)-D-GlcNAc (2) and alpha-L-Fuc-(1-->6)-beta-D-Gal-(1-->4)-D-GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl alpha-L-fucopyranoside and beta-D-Gal-(1-->4)-D-GlcNAc. The enzyme formed the trisaccharides 1-3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. alpha-L-fucosidase led to the regioselective synthesis of trisaccharides containing a (1-->3)-linked alpha-L-fucosyl residue. When beta-D-Gal-(1-->4)-D-GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and alpha-L-Fuc-(1-->3)-beta-D-Gal-(1-->4)-D-Glc (3'-O-alpha-L-fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Enzymatic synthesis of β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2-p as a carbohydrate unit of mucin-type 2 core

We have established a synthetic method for obtaining β-D-Gal-(1→3)-[β-D-GlcNAc-(1→6)]-α-D-GalNAc-OC6H4NO2 -p (1), which is a carbohydrate unit of mucin-type 2 core. A β-N-acetyl-D-hexosaminidase from Nocardia orientalis catalyzed the synthesis of the desired compound 1 with its isomers β-D-GalNAc-(1→6)-β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p (2) β-D-GlcNAc-(1→3)-β-D-Glc-(1→3)-α-D-GalNAc-OC6H4NO2-p (3) through N-acetylglucosaminyl transfer from N,N′-diacetylchitobiose and β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p. The enzyme formed the trisaccharides 1, 2, and 3 in 14% overall yield based on β-D-Gal-(1→3)-α-D-GalNAc-OC6H4NO2-p as an acceptor substrate, and in the ratio of 44:32:24. In this way, N-acetylglucosaminyl transfer favored O-6 of the acceptor rather than O-6′, and occurred to a lesser extent at O-3′. This reaction was efficient enough to allow a one-pot preparation of the desired carbohydrate unit of mucin-type 2 core. When β-D-Gal-(1→3)-β-D-GalNAc-OC6H4NO2-p was used as an acceptor, the enzyme also synthesized three kinds of trisaccharides in the same regioselectivity with respect to O-6 and O-6′ versus O-3′ of the acceptor.     

Substrate specificity of N-acetylhexosaminidase from Aspergillus oryzae to artificial glycosyl acceptors having various substituents at the reducing ends

The substrate specificity of N-acetylhexosaminidase (E.C. 3.2.1.51) from Aspergillus oryzae was examined using p-nitrophenyl 6-O-sulfo-N-acetyl-beta-D-glucosaminide (6-O-sulfo-GlcNAc-O-pNP) as the glycosyl donor and a series of beta-d-glucopyranosides and N-acetyl-beta-D-glucosaminides with variable aglycons at the anomeric positions as the acceptors. When beta-D-glucopyranosides with methyl (CH(3)), allyl (CH(2)CHCH(2)), and phenyl (C(6)H(5)) groups at the reducing end were used as the acceptors, this enzyme transferred the 6-O-sulfo-GlcNAc moiety in the donor to the location of O-4 in these glycosyl acceptors with a high regioselectivity, producing the corresponding 6-O-sulfo-N-acetylglucosaminyl beta-D-glucopyranosides. However, beta-D-glucopyranose lacking aglycon was a poor substrate for transglycosylation. This A. oryzae enzyme could also accept various N-acetyl-beta-D-glucosaminides carrying hydroxyl (OH), methyl (CH(3)), propyl (CH(2)CH(2)CH(3)), allyl (CH(2)CHCH(2)) and p-nitrophenyl (pNP; C(6)H(4)-NO(2)) groups at their aglycons, yielding 6-O-sulfo-N-acetylglucosaminyl-beta(1-->4)-disaccharide products.     

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.     

The hydrolytic and transferase action of alternanase on oligosaccharides.

Alternanase is an enzyme which endo-hydrolytically cleaves the alpha-(1-->3), alpha-(1-->6)-linked D-glucan, alternan. The main products are isomaltose, alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc and the cyclic tetrasaccharide cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus D-glucose, with panose being converted at a much faster rate. The tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc is hydrolyzed to D-glucose plus the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc. Alternanase does not act on isomaltotriose, theanderose (6(Glc)-O-alpha-D-Glcp sucrose), or alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl alpha-isomaltoside, but not from 4-nitrophenyl alpha-D-glucopyranoside, 4-nitrophenyl alpha-isomaltotrioside, or 4-nitrophenyl alpha-isomaltotetraoside.     

Application of selectively acylated glycosides for the α-galactosidase-catalyzed synthesis of disaccharides

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.     

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.     

Production of a new sucrose derivative by transglycosylation of recombinant Sulfolobus shibatae beta-glycosidase

The gene encoding beta-glycosidase of the hyperthermophilic archaea Sulfolobus shibatae (SSG) was expressed in Escherichia coli. Recombinant SSG (referred to as rSSG hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. When sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by TLC and high performance anion exchange chromatography (HPAEC). The sucrose transglycosylation product was isolated by paper chromatography, and its chemical structure was determined by 1H and 13C NMR. The sucrose transfer product was determined to be beta-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-beta-d-fructofuranoside with a galactose molecule linked to sucrose via a beta-(1-->6)-glycosidic bond.     

Oligomers of glycamino acid

Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH(2)-Glc-beta-CO(2)H 1, 3-NH(2)-Glc-beta-CO(2)H 2, 4-NH(2)-Glc-beta-CO(2)H 3, and 6-NH(2)-Glc-beta-CO(2)H 4 and constructed four types of homo-oligomers, beta(1-->2)-linked I, beta(1-->3)-linked II, beta(1-->4)-linked III, and beta(1-->6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and NMR spectral studies of these oligomers suggested that only the beta(1-->2)-linked homo-oligomer possessed a helical structure that seems to be predetermined by the linkage position. Homo-oligomers with beta(1-->2)-linkages I and beta(1-->6)-linkages IV were also subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with naturally occurring sulfated polysaccharides such as heparin.     

Concise syntheses of arabinogalactans with beta-(1-->6)-linked galactopyranose backbones and alpha-(1-->3)- and alpha-(1-->2)-linked arabinofuranose side chains

4-methoxyphenyl glycosides of 2,3'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl tetraose (16), 3',2'-bis-alpha-L-arabinofuranosyl branched beta-D-(1-->6)-linked galactopyranosyl hexaose (27), and a twentyose (42) consisting of beta-(1-->6)-linked D-galactopyranosyl pentadecaoligosaccharide backbone with alpha-L-arabinofuranosyl side chains alternately attached at C-2 and C-3 of the middle galactose residue of each consecutive beta-(1-->6)-linked galactotriose unit of the backbone, were synthesized with isopropyl 3-O-allyl-2,4-di-O-benzoyl-1-thio-beta-D-galactopyranoside (6), 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (7), 2,3,5-tri-O-benzoyl-alpha-L-arabinofuranosyl trichloroacetimidate (12), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (17), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (19), and 2,6-di-O-acetyl-3,4-di-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate (28) as the key synthons. Condensation of 6 with 7 gave the disaccharide donor 8, and subsequent condensation of 8 with 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->6)-2-O-acetyl-3,4-di-O-benzoyl-beta-D-galactopyranoside (9) followed by selective deacetylation afforded the tetrasaccharide acceptor 11. Coupling of 11 with 12 gave the pentasaccharide 13, its deallylation followed by coupling with 12, and debenzoylation gave the hexasaccharide 16 with beta-(1-->6)-linked galactopyranose backbone and 2- and 3'-linked alpha-L-arabinofuranose side chains. The octasaccharide 27 was similarly synthesized, while the twentyoside 42 was synthesized with tetrasaccharides 33 or 24 as the donors and 23, 36, 38, and 40 as the acceptors by consecutive couplings followed by deacylation.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.     

Interaction of soluble glucosyl- and mannosyl-transferase enzyme activities in the synthesis of a glucomannan

A neutral-detergent-solubilized-enzyme preparation derived from Phaseolus aureus hypocotyls contains two types of glycosyltransferase activity. One, mannosyltransferase enzyme activity, utilizes GDP-alpha-d-mannose as the sugar nucleotide substrate. The other, glucosyltransferase enzyme activity, utilizes GDP-alpha-d-glucose as the sugar nucleotide substrate. The soluble enzyme preparation catalyses the formation of what appears to be a homopolysaccharide when either sugar nucleotide is the only substrate present. A beta-(1-->4)-linked mannan is the only polymeric product when only GDP-alpha-d-mannose is added. A beta-(1-->4)-linked glucan is the only polymeric product when only GDP-alpha-d-glucose is added. In the presence of both sugar nucleotides, however, a beta-(1-->4)-linked glucomannan is formed. There are indications that endogenous sugar donors may be present in the enzyme preparation. There appear to be only two glycosyltransferases in the enzyme preparation, each catalysing the transfer of a different sugar to the same type of acceptor molecule. The glucosyltransferase requires the continual production of mannose-containing acceptor molecules for maintenance of enzyme activity, and is thereby dependent upon the activity of the mannosyltransferase. The mannosyltransferase, on the other hand, does not require the continual production of glucose-containing acceptors for maintenance of enzyme activity, but is severely inhibited by GDP-alpha-P-glucose. These properties promote the synthesis of beta-(1-->4)-linked glucomannan rather than beta-(1-->4)-linked glucan plus beta-(1-->4)-linked mannan when both sugar nucleotide substrates are present.     

Biosynthesis of galactogen: identification of a beta-(1----6)-D-galactosyltransferase in Helix pomatia albumen glands

A beta-(1----6)-D-galactosyltransferase has been purified over 2000-fold by affinity chromatography on UDP-p-aminophenyl-Sepharose. The enzyme, from a pellet fraction (8000 x g) of Helix pomatia albumen gland, catalyzes transfer of D-galactose from UDP-galactose to a (1----6) linkage on acceptor H. pomatia galactogen. Three other polymers served as acceptors: beef lung galactan, Lymnaea stagnalis galactogen and arabinogalactan from larch wood. To determine the linkage specificity of the enzyme, it was incubated with UDP-D-galactose and acceptor galactogen that had been tritiated previously by treatment with galactose oxidase and [3H]KBH4. The [3H]galactogen reaction product was recovered, methylated, hydrolyzed and acetylated; tritiated derivatives were identified by mass spectroscopy of effluent fractions separated by gas chromatography. This analysis revealed that (1----6)-linked galactosyl groups had been added to the enzyme-treated acceptor galactogen. Also identified was a hydrolytic enzyme that removed terminal alpha 1,2-linked L-galactosyl residues from H. pomatia galactogen.     

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis> L.) seedlings

We investigated a galactosyltransferase (GalT) involved in the synthesis of the carbohydrate portion of arabinogalactan-proteins (AGPs), which consist of a beta-(1-->3)-galactan backbone from which consecutive (1-->6)-linked beta-Gal p residues branch off. A membrane preparation from 6-day-old primary roots of radish ( Raphanus sativus L.) transferred [(14)C]Gal from UDP-[(14)C]Gal onto a beta-(1-->3)-galactan exogenous acceptor. The reaction occurred maximally at pH 5.9-6.3 and 30 degrees C in the presence of 15 mM Mn(2+) and 0.75% Triton X-100. The apparent K(m) and V(max) values for UDP-Gal were 0.41 mM and 1,000 pmol min(-1) (mg protein)(-1), respectively. The reaction with beta-(1-->3)-galactan showed a bi-phasic kinetic character with K(m) values of 0.43 and 2.8 mg ml(-1). beta-(1-->3)-Galactooligomers were good acceptors and enzyme activity increased with increasing polymerization of Gal residues. In contrast, the enzyme was less efficient on beta-(1-->6)-oligomers. The transfer reaction for an AGP from radish mature roots was negligible but could be increased by prior enzymatic or chemical removal of alpha- l-arabinofuranose (alpha- l-Ara f) residues or both alpha- l-Ara f residues and (1-->6)-linked beta-Gal side chains. Digestion of radiolabeled products formed from beta-(1-->3)-galactan and the modified AGP with exo-beta-(1-->3)-galactanase released mainly radioactive beta-(1-->6)-galactobiose, indicating that the transfer of [(14)C]Gal occurred preferentially onto consecutive (1-->3)-linked beta-Gal chains through beta-(1-->6)-linkages, resulting in the formation of single branching points. The enzyme produced mainly a branched tetrasaccharide, Galbeta(1-->3)[Galbeta(1-->6)] Galbeta(1-->3)Gal, from beta-(1-->3)-galactotriose by incubation with UDP-Gal, confirming the preferential formation of the branching linkage. Localization of the GalT in the Golgi apparatus was revealed on a sucrose density gradient. The membrane preparation also incorporated [(14)C]Gal into beta-(1-->4)-galactan, indicating that the membranes contained different types of GalT isoform catalyzing the synthesis of different types of galactosidic linkage.