SUBSTRATE SUBSITE RECOGNITION OF THE XYLOGLUCAN ENDO-TRANSGLYCOSYLASEOR XYLOGLUCAN-SPECIFIC ENDO-(1-]4)-BETA-D-GLUCANASE FROM THE COTYLEDONS OF GERMINATED NASTURTIUM (TROPAEOLUM-MAJUS L) SEEDS

We have investigated recognition requirement transglycosylase/xylogluc an-specific endo-(1 --> 4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirel y of the Glc, subunits XXXG, XLXG, XXLG and XLLG, where G represents a n unsubstituted glucose residue, X a xylose-substituted glucose residu e and L a galactosyl-xylose-substituted glucose residue. Thus in the x yloglucan sequence shown below, the xylose (Xyl) residues at the backb one glucose (Glc) residues numbered - 3, - 2, + 2 and + 3 may be galac tose-substituted, and NXET cleaves between the unsubstituted glucose a t - 1 and the xylose-substituted glucose at + 1, which never carries a galactosyl substituent. [GRAPHICS] We have isolated the xyloglucan ol igosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind see d xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to gi ve GXXGXXXG and GLLGXLLG, and have identified and compared the product s of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in n ative tamarind and nasturtium seed xyloglucans with those in NXET dige sts of these polysaccharides. Using these and existing data we have de monstrated that NXET action does not require xylose-substitution at gl ucose residues - 4, - 2, + 1 and + 3 and that xylose substitution at 2, is a requirement. There may also be a requirement for xylose subst itution at - 3. We have demonstrated also that galactosyl substitution of a xylose residue at + 1 prevents, and at - 2 modifies, chain-cleav age. A partial model for the minimum substrate binding requirement of NXET is proposed.