Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction.

Plant xyloglucan endotransglycosylase (XET, EC 2.4.1.207) degrades its substrate by a transglycosylation mechanism while endo-cleaving xyloglucan (XG) molecules at their beta-1,4-linked polyglucosyl main chain and transferring the newly generated reducing chain ends to hydroxyls at C-4 of non-reducing glucosyl ends of the main chains of other XG molecules or of low-Mr XG-fragments (OS). Kinetic data obtained with purified nasturtium seed (Tropaeolum majus, L.) XET while using high-Mr xyloglucan and 3H-labeled XGOS alditols (DP 7-9) as substrates could be best fitted to the model for Ping-Pong Bi Bi reaction mechanism. Such mechanism is typical for transglycosylases operating with retention of the anomeric configuration of the formed glycosidic bond and involving the formation of a covalent glycosyl-enzyme reaction intermediate.

The glycoprotein character of multiple forms of Aspergillus polygalacturonase.

Comparisons of known primary structures of polygalacturonases show that extent and localization of potential N-glycosylation sites differ. Some sites are similar in position and adjacent to strictly conserved residues at the potential active site. The presence of N-acetylglucosamine and mannose in the molecules of two homogeneous, major Aspergillus sp. polygalacturonase forms was confirmed by IR spectroscopy. The purification method, based on interaction of the carbohydrate part with concanavalin A immobilized on chlorotriazine bead cellulose, was optimized. Deglycosylation with N-glycosidase F under denaturating and nondenaturating conditions led to molecular mass decreases followed by complete inactivation of the polygalacturonase enzyme activity. These results show the importance of glycosylation in these protein forms, while the comparative patterns establish both variability and some similarities in overall glycosylation architectures.

An essential tyrosine residue of Aspergillus polygalacturonase.

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

Pectinase Aspergillus sp. polygalacturonase: multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases.

Nine forms of Aspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with free alpha-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from other Aspergillus species (72-75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66-71% residue differences toward the Erwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties.

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides.

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.