The role of the glucuronoxylan carboxyl groups in the action of endoxylanases of three glycoside hydrolase families: A study with two substrate mutants.

BACKGROUND: Bacterial appendage-dependent GH30 glucuronoxylan hydrolases recognize the substrate through an ionic interaction of a conserved positively charged arginine with the carboxyl group of 4-O-methyl-d-glucuronic acid. One of the options to verify this interaction is preparation of enzyme mutants. An alternative approach is a chemical modification of the substrate, glucuronoxylan, in which the free carboxyl group in all residues of MeGlcA is eliminated. METHODS: In this work the carboxyl groups of 4-O-methyl-d-glucuronic acid residues of an alkali extracted beechwood xylan were esterified with methanol. A water-soluble fraction of the polysaccharide methyl ester was converted by NaBH4 reduction to the second soluble derivative, 4-O-methylglucoxylan. Specific activities of several endoxylanases (EXs) of GH families 10, 11 and 30 were determined on glucuronoxylan, and its two new uncharged derivatives. RESULTS: Elimination of the free carboxyl group from the polysaccharide did not influence activities of GH10 EXs, but resulted in 50% decrease of specific activity of GH11 EXs, and led to more than 300-fold reduction of specific activity of Erwinia chrysanthemi GH30 xylanase. CONCLUSIONS: These results confirm the crucial role of the interactions between GH30 xylanases and the MeGlcA carboxyl group for efficient cleavage of the polysaccharide. Analysis of the hydrolysis products by TLC and MS confirmed that all three types of xylanases hydrolyzed uncharged glucuronoxylans similarly as the original one. SIGNIFICANCE: The uncharged glucuronoxylan derivatives will be useful to differentiate GH30 xylanases with various degree of selectivity for glucuronoxylan, including fungal enzymes without the conserved arginine.

Xyloglucan degradation using different radiation sources: a comparative study.

Tamarind seed xyloglucan was subjected to different radiation sources-ultrasound, gamma-radiation, and microwave heating, and the effects of these energies upon its molecular and structural properties were characterised by gel permeation chromatography, viscometry, sugar analysis, FT-IR and NMR spectroscopic techniques. In dependence on the degradation methods and experimental conditions used, the decrease of the relative molecular mass (RMM) was accompanied with alteration of the primary structure. Depolymerisation by ultrasound at a frequency of 20 kHz yielded after 120 min products with RMM of about 131 x 10(3) without significant alteration of the primary structure of the polysaccharide. Intense degradation of XG started by microwave heating at pH 1.5 yielding polymers with RMM in the range of higher oligosaccharides, however, with changed sugar composition due to cleavage of the glycosyl side chains. At gamma-irradiation doses >40 kGy, next to chain cleavage, very high-molecular mass components exhibiting UV(254)-absorption were formed, and the RMM decreased to about 50 x 10(3) at the highest applied dose (100 kGy). The results of the comparative study suggest that ultrasonication was the most convenient procedure to decrease the RMM of xyloglucan to 130 x 10(3) and preserve the primary structure of the polysaccharide.

(4-O-Methyl-alpha-D-glucurono)-D-xylan from Rudbeckia fulgida, var. sullivantii (Boynton et Beadle).

From the medicinal plant Rudbeckia fulgida, var. sillivantii (Boynton et Beadle) a low-molecular-mass (4-O-methyl-alpha-D-glucurono)-D-xylan was isolated by alkaline extraction, followed by ethanol precipitation, ion-exchange chromatography and gel filtration. The results of compositional and linkage analyses, supported by those of 1H and 13C NMR measurements of oligomers generated on partial acid hydrolysis, showed the (1-->4)-linked beta-D-xylopyranosyl backbone with about 18% of 4-O-methyl-D-glucuronic acid attached to O-2 of the xylose residues. From the mean distance of adjacent carboxyl groups, obtained from experimentally determined single-ion activity coefficients of calcium counterions, it followed that the uronic acid units are separated and distributed regularly along the xylan chain, i.e. approximately each sixth D-xylose unit bears a 4-O-methyl-D-glucuronic acid residue.

Conformations of (1----4)-linked alpha-D-galacturono-di- and -tri-saccharides in solution analysed by n.m.r. measurements and theoretical calculations.

The conformations of the (1----4)-linked alpha-D-galacturono-di- (1) and -tri-saccharide (2) in aqueous solutions have been analysed by n.m.r. spectroscopy and MM2CARB calculations. The 3JC.H. and n.O.e. values did not change with temperature and were comparable for 1 and 2. Four energy regions were found on the relaxed (phi, psi) map for 1 computed by the MM2CARB method. Theoretical n.O.e. values, based on the geometry and the abundance of the most populated conformer, accorded with experimental values. The magnitudes of phi H and psi H for the glycosidic bond suggest that a right-handed three-fold helical arrangement can be formed by pectic acid oligosaccharides in solution.

Immunomodulatory activity of acidic xylans in relation to their structural and molecular properties.

The structure/function relationship of two acidic heteroxylan types, the arabino-(glucurono)xylan from corn cobs (AGX) and 4-O-methylglucuronoxylans (GXs) from beechwood and three medicinal herbs (Rudbeckia, Altheae, and Mahonia), has been studied. The effect of the molecular mass of AGX, as well as the content and distribution of the 4-O-methylglucuronic acid side chains in GXs on the immunological activity of these xylans was characterized by their biological response in the mitogenic and comitogenic thymocyte in vitro tests. Depolymerization of AGX by ultrasonication resulted in unequivocal decrease of the immunomodulatory activity, whereas already a short treatment by endo-beta-1,4-xylanase brought about a significant increase in its activity when applied in the highest dose. In the case of the GX samples, neither the uronic acid content nor the distribution pattern of the uronic acid side chains was found to be determinant for the expression of their immunomodulatory activity.

Study of the interactions of D- and L-polylysine enantiomers withpectate in aqueous solutions.

The interaction between D- and L-enantiomers of polylysine and potassium pectate was studied by means of CD, microcalorimetry, and osmometry. Upon binding with pectate, only poly(L-lysine) undergoes a coil to alpha-helix transition, while poly(D-lysine) remains in the disordered state. This suggest that the energetics of the interaction is influenced by stereochemical constraints besides electrostatic forces. Experimental findings from microcalorimetry suggest that a contribution to the overall enthalpy of binding comes from the polysaccharidic moiety. Stoichiometry of the macromolecular complexes studied by osmometry gives a polylysine:pectate ratio of 3:1, in agreement with the respective degree of polymerization of the two polyelectrolytes.

Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation.

The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent.