Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Characterization of a long-chain α-galactosidase from Papiliotrema flavescens.

α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and ß-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.

A novel fluorescence assay and catalytic properties of Crh1 and Crh2 yeast cell wall transglycosylases.

The mechanical properties of fungal cell walls are largely determined by composition and mutual cross-linking of their macromolecular components. Previous work showed that the Crh proteins are required for the formation of cross-links between chitin and glucan at the Saccharomyces cerevisiae cell wall. In the present study, the proteins encoded by CRH1 and CRH2 were heterologously expressed in Pichia pastoris and a sensitive fluorescence in vitro soluble assay was devised for determination of their transglycosylating activities. Both proteins act as chitin transglycosylases; they use soluble chitin derivatives, such as carboxymethyl chitin, glycol-chitin and/or N-acetyl chito-oligosaccharides of DP (degree of polymerization)≥5 as the oligoglycosyl donors, and oligosaccharides derived from chitin, ß-(1,3)-glucan (laminarin) and ß-(1,6)-glucan (pustulan), fluorescently labelled with sulforhodamine or FITC as acceptors. The minimal number of intact hexopyranose units required by Crh1 and/or Crh2 in the molecule of the acceptor oligosaccharide was two and the effectivity of the acceptor increased with the increasing length of its oligosaccharide chain. Products of the transglycosylation reactions were hybrid molecules composed of the acceptor and portions of carboxymethyl chitin attached to its non-reducing end. Both proteins exhibited a weak chitinolytic activity in different assays whereby the ratio of endo- compared with exo-chitinase activity was approximately 4-fold higher in Crh1 than in Crh2. The pH optimum of both enzymes was 3.5 and the optimum temperature was 37°C. The results obtained in vitro with different fluorescently labelled oligosaccharides as artificial chitin acceptors corroborated well with those observed in vivo.