Substrate subsite recognition of the xyloglucan endo-transglycosylase or xyloglucan-specific endo-(1?4)-?-d-glucanase from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds

Abstract

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(1-->4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered -3, -2, +2 and +3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. [formula: see text] We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose-substitution at glucose residues -4, -2, +1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.

A xyloglucan oligosaccharide‐active, transglycosylating‐D‐glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings – purification, properties and characterization of a cDNA clone

“…The first three have been isolated and characterized as a xyloglucan-specific endo-(1→4)-β-D-glucanase or XET (Edwards et al, 1986;Fanutti et al, 1993Fanutti et al, , 1996de Silva et al, 1993), a xyloglucan-active β-galactosidase Reid et al, 1988) and a xyloglucan oligosaccharide-specific α-xylosidase . We have now purified the major β-glucosidase activity from nasturtium cotyledons undergoing massive mobilization of xyloglucan from the storage cell walls, and have sequenced the corresponding cDNA.…”

“…Of central importance is the endo-depolymerizing action of nasturtium XET (NXET) which catalyses both hydrolysis (Edwards et al, 1986) and endo-transglycosylation of storage xyloglucans, with chain cleavage occurring between the structural subunits. This cleavage specificity reflects both the substrate recognition requirement of the enzyme and the regular structure of the polymer (Fanutti et al, 1996). In vitro, the hydrolytic action of NXET is evident only when the concentration of specific acceptor chain ends (non-reducing ends of xyloglucans or xyloglucan oligosaccharides) is low .…”

“…The storage xyloglucans from seeds are closely similar in their structural features to the hemicellulosic xyloglucans of plant primary cell walls (Hayashi, 1989), but the seed polymers are not fucosylated. Recent studies of seed xyloglucans show that they consist almost entirely of the structural subunits XXXG, XXLG, XLXG and XLLG, although in varying proportions (Buckeridge et al, 1992;Fanutti et al, 1996;Gidley et al, 1991;York et al, 1990) according to the botanical source. Unlike the primary cell wall xyloglucans, seed xyloglucans are very easily extracted from the plant tissues with hot water (Reid, 1985).…”

Lack of α-Xylosidase Activity in Arabidopsis Alters Xyloglucan Composition and Results in Growth Defects

“…There is also some evidence that a-xylosidase could attack the nonreducing ends of polymeric xyloglucan. A Tropaeolum a-xylosidase can remove Xyl from oligosaccharides with a backbone of at least eight Glc residues (Fanutti et al, 1991(Fanutti et al, , 1996. Additionally, the presence of up to 17% Xyl-deficient subunits, such as GXXG, exclusively in the enzyme-accessible fraction of pea xyloglucan has been attributed to a-xylosidase activity at the nonreducing ends of the polymer (Guillén et al, 1995;Pauly et al, 2001).…”

Structural Evidence for the Evolution of Xyloglucanase Activity from Xyloglucan Endo-Transglycosylases: Biological Implications for Cell Wall Metabolism

“…The three-dimensional structure of this enzyme has been solved (Johansson et al, 2004), and the catalytic mechanism has been probed using a range of synthetic xylogluco-oligosaccharides to elucidate details of enzymesubstrate interactions . Intriguingly, xyloglucanase activity and gene sequence have been conclusively linked for only one member of GH16, Tm-NXG1 from Tropaeolum majus (nasturtium), which is a predominant endo-xyloglucanase that can also perform xyloglucan endo-transglycosylation at elevated concentrations of acceptor substrates (Edwards et al, 1986;de Silva et al, 1993;Fanutti et al, 1993Fanutti et al, , 1996Chanliaud et al, 2004). (A) The chemical mechanism.…”