Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 306024712, USA
Received on June 1, 1999; revised on August 13, 1999; accepted on August 19, 1999.
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Abstract |
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Key words: endoglucanase/transglycosylation/ß-glycoside/xyloglucan/cellulose
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Introduction |
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The transglycosylation of polysaccharides is a biologically important process in vivo. For example, xyloglucan (XG) endotransglycosylases cleave and religate the polysaccharides that cross-link the walls surrounding rapidly expanding cells in flowering plants (Fry et al., 1992). Transglycosylation reactions have also been widely used to prepare glycosides in vitro (Crout and Vic, 1998
; McCleary, 1999
). Typically an exoglycosidase is incubated with a low molecular weight donor substrate, such as an aryl glycoside or a glycosyl halide, and a suitable acceptor substrate. Recently, the capacity of an endoglycosidase to catalyze the in vitro transfer of a decasaccharide from a naturally occurring glycopeptide to a synthetic N-acetylglucosylaminyl peptide acceptor substrate has been demonstrated (Yamomoto et al., 1998
). Endoglucanases (EGs) have also been used to catalyze transglycosylation reactions in which the donor substrate is a diglycosyl halide (e.g., lactosyl fluoride) and the acceptor substrate is another oligosaccharide (Moreau and Driguez, 1996
; Armand et al., 1997
). However, endoglycosidases have not previously been used to prepare glycosides via transglycosylation reactions in which a polysaccharide is used as the donor substrate.
Glycosyl hydrolases are commonly used to catalyze the formation of a glycosidic bond between two sugars, that is, via reactions in which the acceptor substrate is itself a carbohydrate. However, the products of reactions in which the acceptor substrate is a small alcohol are also extremely useful. For example, allyl glycosides are versatile reagents for the chemical synthesis of complex carbohydrate structures. Isomerization of the allyl aglycon generates a highly reactive vinyl glycoside (Marra et al., 1992; Boons and Isles, 1996
), which can be used as a reagent in chemically catalyzed glycosylation reactions. Hydrolase-catalyzed transglycosylation and reverse-hydrolysis reactions have been used (Vic and Crout, 1995
; Gibson et al., 1997
) to produce allyl and butenyl glycosides for this purposes, but the transglycosylation products produced thus far have consisted of only one or two sugars and the reactive aglycon.
This paper describes the first use of a polysaccharide as a donor substrate for a preparative glycosyltransferase reaction. The use of polysaccharide donor substrates has great potential for the rapid and economic production of useful glycosides. Polysaccharides produced by plants and microbes represent a wide range of structural motifs and are often easy to prepare in large quantities. For example, the bacterial polysaccharide xanthan and the plant polysaccharide cellulose are routinely produced by the ton.
Another important class of polysaccharides are the hemicellulosic XGs, which are major constituents of the cell walls of higher plants (York et al., 1990, 1993, 1996). The XG backbone is composed of ß-(1
4)-linked D-Glcp residues, up to 75% of which are branched, bearing a sidechain at O-6 (Figure 1). The structures of the XG sidechains vary in different plant tissues and species (York et al., 1990
, 1996), the most common being: (1)
-D-Xylp-; (2) ß-D-Galp-(1
2)-
-D-Xylp-; (3)
-L-Fucp-(1
2)-ß-D-Galp-(1
2)-
-D-Xylp-; and (4)
-L-Araf-(1
2)-
-D-Xylp-. EGs from several sources selectively hydrolyze the unbranched ß-(1
4)-linked D-Glcp residues in the backbone of the XG, generating a mixture of biologically active xyloglucan oligosaccharides (XGOs, Figure 1) (York et al., 1984
). This report describes EG-catalyzed transglycosylation reactions in which the donor substrate is either cellulose or XG. These reactions can be used to prepare relatively large and complex oligoglycoside products that may be useful in themselves or may be used as synthons for the production of more complex structures by chemical methods.
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Results |
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Several other alcohols were screened for their suitability as acceptor substrates for the EG-catalyzed glycosyl transfer reaction (Table I). The MALDI-TOF mass spectra of the products obtained when tamarind XG was treated in the presence of various alcohols are shown in Figure 2. Some of the alcohols, most notably those containing a sulfhydryl group, inhibit the catalytic activity of the EG, and no depolymerization products are observed in the mass spectra of the treated material. Isopropanol, a secondary alcohol, appeared to be a much poorer acceptor substrate than the homologous primary alcohol n-propanol. However, trans-1,2-cyclohexanediol, which contains only secondary alcohols, is an efficient acceptor substrate; (1R,2R)-trans-1,2-cyclohexanediol, in which the oxygen-bearing carbons have the same absolute stereochemistry as C3 and C4 of a glucopyranosyl residue, is a better acceptor than the 1S,2S isomer. Saligenin (o-hydroxybenzyl alcohol) is not soluble in H2O at a concentration of 20%, and so this acceptor was included in the reaction mixture as a suspension. Nevertheless, ~25% of the products generated under these conditions were saligenin glycosides. It was not determined which of the two hydroxyl groups in saligenin was more reactive.
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The ß-Glcp residue of XLLG-gro bearing the glyceryl aglycon was identified by a long-range heteronuclear coupling from its anomeric carbon (C1 103.0) to two geminal protons (H1,
3.759 and H1'
3.913) of the glycerol moiety. This assignment was confirmed by the observation of NOESY crosspeaks between H1 (
4.504) of this ß-Glcp residue and the same two geminal protons. These results indicate that the transglycosylation product is a ß-glycoside linked to one of the primary hydroxyl groups of the glycerol moiety, as expected, based on the greater reactivity of n-propanol relative to iso-propanol (see above). The absolute stereochemistry of the glycerol (sn-1-linked or sn-3-linked) in this major product was not determined.
Preparation of ß-methyl cellobiosides from cellulose
EG-treatment of cellulose in the presence of methanol as an acceptor substrate resulted in the generation of cellobiose, glucose, ß-methyl cellobioside, and ß-methyl glucoside. The EG-catalyzed depolymerization of insoluble cellulose is a heterogeneous reaction, and only a small proportion (usually less than 5%) of the cellulose was solubilized under the conditions used. However, up to 50% of the soluble products were recovered as ß-glycosides. Reconstituted, hydrated cellulose (i.e., dialysis tubing), appeared to be a better substrate than crystalline cellulose. EG-treatment of Sigmacell 100 generated several unidentified products in addition to cellobiose, glucose, ß-cellobiosides, and ß-glucosides, which were the dominant products generated when Avicel or dialysis tubing was used as a substrate. The presence of 20% methanol did not significantly inhibit the capacity of the EG to solubilize cellulose, and yields were not linearly dependent on the amount of EG or cellulose used in the reaction (Table IV). In general, the absolute amount of EG-solubilized carbohydrate increased ~4-fold when the amount of cellulose substrate was increased 10-fold (all other conditions being kept equal). The absolute amount of EG-solubilized carbohydrate increased ~2-fold when the amount of EG was increased 10-fold. As cellulose is much less expensive than EG, the most cost-effective implementations of this reaction will likely involve conditions that solubilize only a small portion of the cellulose. Pretreatment of the cellulose with a swelling agent to make it more accessible to the enzyme may also be advantageous.
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Discussion |
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It is, at first glance, somewhat surprising that the ß-glycosides generated by EG-catalyzed transglycosylation reactions are resistant to hydrolysis by the EG (Crout and Vic, 1998), as it is often postulated that enzymes catalyze the forward and reverse reactions with equal efficiency and that the distribution of products depends on their relative thermodynamic stabilities.
The resistance of the ß-glycosides to EG-catalyzed hydrolysis is consistent with the catalytic mechanism proposed (Sulzenbacher et al., 1996) for Family 7 EGs (Figure 5), such as the Megazyme endoglucanase (EG-1 from Trichoderma reesei) used in this study. According to this model, binding of a substrate distorts the geometry of the glucosyl residue in the 1 subsite, placing the potential leaving group in the axial orientation, consistent with the proposed geometry of the transition state. The distorted axial geometry of the glycosidic oxygen is energetically unstable in aqueous solution, where the glycosidic oxygens of ß-glucosyl residues are invariably found in the equatorial orientation. Therefore, the increase in free energy due to the distortion must be compensated by a decrease in free energy due to the association of the glucan substrate with the EG. Presumably, the distortion of the glucosyl residue in the -1 subsite of the EG depends, in part, on the strong interaction of the adjacent glucosyl residue with the +1 binding subsite of the EG, leading to a strained complex whose total free energy is lower than that of the isolated enzyme and substrate (Sulzenbacher et al., 1996
). Stabilization of the transition state facilitates the formation of a enzyme substrate intermediate in which the glucosyl residue is covalently attached to the sidechain of a glutamic acid. During hydrolysis reactions, this intermediate is attacked by a water molecule, releasing the product as an oligosaccharide whose reducing glucose residue is in the ß-configuration (i.e., with retention of anomeric configuration). During transglycosylation reactions, the intermediate is attacked by an alcohol, releasing the product as a ß-glycoside. The resistance of the alkyl ß-glycosides to hydrolysis by the EG is consistent with this scenario, in that the aglycon moieties of these glycosides are chemically distinct from the glucosyl residues that have a putatively strong interaction with the +1 binding subsite of the EG. Apparently, the weak interaction of the alkyl aglycon with the +1 binding subsite does not provide sufficient energy to induce geometric distortion of the glucosyl residue in the -1 subsite. Therefore, no productive complex is formed, even if the alkyl glycoside happens to bind to the active site of the EG and the alkyl glycoside is not a substrate for the EG.
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Conclusion |
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Materials and methods |
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SEC
Samples were desalted by concentrating them to 25 ml and applying them to a column (2.5 x 45 cm) of Sephadex G-10 (Pharmacia) eluted with deionized water (1 ml/min) The anthrone assay (Dische, 1962) was used to determine the carbohydrate content of the collected fractions (2 ml), and the conductivity of the fractions was measured (model 1052A conductometer, Amber Science) to determine their salt content. Oligoglycosides were separated by SEC on Bio-Gel P chromatography supports (Bio-Rad Laboratories). Samples (2 ml) were injected onto a Bio-Gel P-10 column (1.6 x 75 cm) or two Bio-Gel P-2 columns (1.6 x 80 cm) connected in series, and eluted with deionized water (0.10.2 ml/min). The carbohydrate content of the collected fractions (2 ml) was determined by the anthrone assay (Dische, 1962
).
Reversed-phase chromatography
Oligoglycoside samples were dissolved in water, and up to 250 µl was injected onto a "semi-preparative" octadecyl silica column (10 x 250 mm, Lichrosorb RP-18, Merck). The oligoglycosides were eluted (3 ml/min) with a linear gradient of aqueous methanol (612% v/v over 60 min) delivered with an HPLC gradient module (Bio-Rad Laboratories) controlled by a Bio-Rad model 700 chromatography workstation. Approximately 5% of the eluent was diverted via a T-splitter to an evaporative light scattering detector (SEDEX 55 ELSD, S.E.D.E.R.E., Alfortville, France), and the remaining 95% was collected manually (Pauly and York, 1998).
ß-Alkyl oligoglycosides from XG
XG, from defatted tamarind seed powder (York et al., 1990), was dissolved (1 mg/ml) in NaOAc buffer (50 mM, pH 5) containing various alcohols (20% v/v) and treated with EG (0.4 units/mg of XG). The solution was incubated (23°C, 24 h) and then boiled (15 min) to deactivate the enzyme. Volatile alcohols, when present, were removed by evaporation under reduced pressure. The products were concentrated to a small volume (<3 ml) and desalted by SEC on Sephadex G-10. XGOs with molecular weights less than 2000 (typically 4090% of the soluble products) were isolated from the desalted product by SEC on Bio-Gel P-10 (data not shown). Oligoglycosides in this low molecular weight fraction were separated by SEC on Bio-Gel P-2 (Figure 3). Individual P-2 fractions (Figure 1) derived from the material generated by EG-treatment of XG in 20% methanol were collected, concentrated, and subjected to reversed-phase HPLC on octadecyl silica, to yield pure ß-methyl oligoglycosides (Figure 4CF).
ß-Alkyl oligoglycosides from cellulose
Reconstituted cellulose (Spectra-Por 6 dialysis tubing (Spectrum), cut into small pieces) was suspended (3.5 g in 200 ml of 50 mM NaOAc buffer, pH 5) containing methanol (20% v/v). EG (10 units) was added and the suspension was incubated (23°C, 24 h). The addition of EG and incubation were repeated two times, after which the reaction mixture was boiled (30 min) to deactivate the enzyme. Methanol was removed by evaporation under reduced pressure. Insoluble material (i.e., undigested cellulose) was then removed by centrifugation (10,000 x g, 20 min, Beckman JA-10 rotor). The soluble products were concentrated to a small volume (5 ml) and desalted by SEC on Sephadex G-10. ß-Methyl cellobioside and ß-methyl-glucoside were isolated from the desalted material by SEC on Bio-Gel P-2.
MALDI-TOF mass spectrometry
MALDI-TOF mass spectra were recorded using a Hewlett Packard G2025A LD-TOF mass spectrometer operating at an accelerating voltage of 4.75 kV and a source pressure of approximately 3 x 107 torr. Desalted, aqueous samples (1 µl) were mixed with a solution (1 µl) of the ionization matrix (1:1 (v/v) 2,5-dihydroxy benzoic acid (0.2 M in 50% aq. CH3CN) and 1-hydroxy isoquinoline (0.06 M in 50% aq. CH3CN)). Analyte and matrix were cocrystallized on the probe by evaporation of the solvent under vacuum. Desorption/ionization was accomplished with a 3 ns pulse ( = 337 nm) from a nitrogen laser.
NMR spectroscopy
ß-Glycosides (15 mg) were dissolved in D2O (1 ml, 99.6 atom % 2H, Cambridge Isotope Laboratories, CIL) and lyophilized to replace exchangeable protons with deuterons. The residues were redissolved in D2O (99.96% atom % 2H, CIL) and NMR spectra were recorded at 298 K with either a Varian Mercury (300 MHz) or Varian Inova (600 MHz or 800 MHz) spectrometer. Double-quantum filtered COSY (Rance et al., 1983), TOSCY (Bax and Davis, 1985
), NOESY (Macura and Ernst, 1980
), HSQC (Bodenhausen and Ruben, 1980
; Norwood et al., 1990
), and HMBC (Bax and Summers, 1986
) experiments were recorded at 600 MHz (and 800 MHz for XLLG-gro). Pulsed-field gradients were used for coherence selection during the HSQC and HMBC experiments. Chemical shifts are reported relative to internal acetone (1H
2.225, 13C
33.0).
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Acknowledgments |
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Footnotes |
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References |
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Bax,A. and Davis,D.G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson., 65, 355360.[ISI]
Bax,A. and Summers,M.F. (1986) 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc., 108, 20932094.[ISI]
Bodenhausen,G. and Ruben,D.J. (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett., 69, 185189.[ISI]
Boons,G.-J. and Isles,S. (1996) Vinyl glycosides in oligosaccharide synthesis. 2. The use of allyl and vinyl glycosides in oligosaccharide synthesis. J. Org. Chem., 61, 42624271.[ISI][Medline]
Carbohydrate Research. (1998) 305.
Crout,D.H.G. and Vic,G. (1998) Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Curr. Opin. Struct. Biol., 98111.
Díaz,A., Fragoso,A., Cao,R. and Vérez,V. (1998) Synthesis and SOD-like activiry of monosaccharide derived thiosemicrbazones. J. Carbohydr. Chem., 17, 293303.[ISI]
Davies,G. and Henrissat,B. (1995) Structures and mechanisms of glycosyl hydrolases. Structure, 3, 853859.[ISI][Medline]
Dische,Z. (1962) Color reactions of carbohydrates. In Whistler,R.L. and Wolfrom,M.L. (eds.), Methods in Carbohydrate Chemistry, Volume 1. Academic Press, New York, pp. 478481.
Fry,S.C., Smith,R.C., Renwick,K.F., Martin,D.J., Hodge,S.K. and Matthews,K.J. (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem. J., 282, 821828.[ISI][Medline]
Fry,S.C., York,W.S., Albersheim,P., Darvill,A.G., Hayashi,T., Joseleau,J.-P., Kato,Y., Lorences,E.P., Maclachlan,G.A., McNeil,M., Mort,A.J., Reid,J.S.G., Seitz,H.U., Selvendran,R.R., Voragen,A.G.J. and White,A.R. (1993) An unambiguous nomenclature for xyloglucan-derived oligosaccharides. Physiol. Plant., 89, 13.[ISI]
Gibson,R.R., Dickinson,R.P. and Boons,G.-J. (1997) Vinyl glycosides in oligosaccharide synthesis. 4. Glycosidase-catalyzed proparation of substituted allyl glycosides. J. Chem. Soc. Perkin Trans., 1, 3360.
Kooiman,P. (1961) The constitution of Tamarindus-amyloid. Rec. Trav. Chim., 80, 849865.[ISI]
Macura,S. and Ernst,R.R. (1980) Elucidation of cross relaxation in liquids by two-dimensional N.M.R. spectroscopy. Mol. Phys., 41, 95117.[ISI]
Marra,A., Esnault,J., Veyrières,A. and Sinaÿ,P. (1992) Isopropenyl glycosides and congeners as novel classes of glycosyl donors: theme and variations. J. Am. Chem. Soc., 114, 63546360.[ISI]
McCleary,B. (1999) Personal communication.
Moreau,V. and Driguez,H. (1996) Enzymatic-synthesis of hemithiocellodextrins. J. Chem. Soc. Perkin Trans., 1, 525527.
Norwood,T.J., Boyd,J., Heritage,J.E., Soffe,N. and Campbell,I.D. (1990) Comparison of techniques for 1H-detected heteronuclear 1H- 15N spectroscopy. J. Magn. Reson., 87, 488501.[ISI]
Pauly,M. and York,W.S. (1998) Isolation of complex carbohydrates by reversed-phase chromatography with evaporative light scattering detection. Am. Biotechnol. Lab., 1998, 1416.
Pauly,M. Andersen,L.N., Kauppinen,S., Kofod,V., York,W.S., Albersheim,P. and Darvill,A.G. (1999) A xyloglucan-specific endo-ß-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme. Glycobiology, 9, 93100.
Rance,M., Sorensen,O.W., Bodenhausen,G., Wagner,G., Ernst,R.R. and Wüthrich,K. (1983) Improved spectral resolution in COSY 1H NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun., 117, 479485.[ISI][Medline]
Sulzenbacher,G., Driguez,H., Henrissat,B., Schülein,M. and Davies,G.J. (1996) Structure of the Fusarium oxysporum endoglucanase I with a nonhydrolyzable substrate analogue: Substrate distortion gives rise to the preferred axial orientation for the leaving group. Biochemistry, 35, 1528015287.[ISI][Medline]
Vic,G. and Crout,D.H.G. (1995) Synthesis of allyl and benzyl ß-D-glucopyranosides and allyl ß-D-galactopyranoside from D-glucose of D-galactose and the corresponding alcohol using almond ß-D-glucosidase. Carbohydr. Res., 279, 315319.[ISI]
Yamomoto,K., Fujimori,K., Haneda,K., Mizuno,M., Inaza,T. and Kuagai,H. (1998) Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase. Carbohydr. Res., 305, 415422.[ISI]
York,W.S., Darvill,A.G. and Albersheim,P. (1984) Inhibition of 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments by a xyloglucan oligosaccharide. Plant Physiol., 75, 295297.[ISI]
York,W.S., van Halbeek,H., Darvill,A.G. and Albersheim,P. (1990) Structure of plant cell walls. 29. Structural analysis of xyloglucan oligosaccharides by 1H-NMR spectroscopy and fast atom bombardment mass spectrometry. Carbohydr. Res., 200, 931.[ISI][Medline]
York,W.S., Harvey,L.K., Guillen,R., Albersheim,P. and Darvill,A.G. (1993) Structural analysis of tamarind seed xyloglucan oligosaccharides using ß-galactosidase digestion and spectroscopic methods. Carbohydr. Res., 248, 285301.[ISI][Medline]
York,W.S., Kolli,V.S.K., Orlando,R., Albersheim,P. and Darvill,A.G. (1996) The structures of arabinoxyloglucans produced by solanaceous plants. Carbohydr. Res., 285, 99128.[ISI][Medline]