Abstract
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.
Abstract
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)-β-d-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-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)-β-d-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-d-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-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.
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Abbreviations
- 2GalManO6-OS:
-
2-galacto-manno-hexasaccharide
- Ara-OS6:
-
Arabino-heptasaccharide
- AraGal-OS:
-
Arabino-galacto-oligosaccharides
- AraXyl-OS:
-
Arabino-xylo-oligosaccharides
- Cello-OS:
-
Cello-oligosaccharides
- Cello-OS3:
-
Cello-triose
- Cello-OS4:
-
Cello-tetraose
- Cello-OS5:
-
Cello-pentaose
- Cello-OS6:
-
Cello-hexaose
- CMC:
-
Carboxymethyl cellulose
- cpk:
-
Atomic colour scheme
- C’XET:
-
TmXET6.3 without putative signal peptide
- GalUA-OS5:
-
Penta-galacturonic acid oligosaccharide
- GalMan-OS:
-
Galacto-manno-oligosaccharides
- GlcMan-OS:
-
Gluco-manno-oligosaccharides
- GH16:
-
Family 16 glycoside hydrolase
- HEC:
-
Hydroxyethyl cellulose
- HPLC:
-
High performance liquid chromatography
- La:
-
Laminarin
- La-OS:
-
Laminari-oligosaccharides
- Man-OS:
-
Manno-oligosaccharides
- Man-OS6:
-
Manno-hexa-oligosaccharide
- MLG-OS:
-
Mixed-linkage (1,3;1,4)-β-d-gluco-saccharides
- MLG-OS4 A, B, C:
-
(1,3;1,4)-β-d-tetra-glucosaccharides A, B, C
- MALDI:
-
Matrix-assisted laser desorption/ionisation
- MS:
-
Mass spectrometry
- OS:
-
Oligosaccharide(s)
- Pu:
-
Pustulan
- Pu-OS:
-
Pustulo-oligosaccharides
- TmXET6.3:
-
Tropaeolum majus XET6.3
- RACE:
-
Rapid Amplification of cDNA Ends
- RMSD:
-
Root-mean square deviation
- SDS-PAGE:
-
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- SR:
-
Sulforhodamine
- TOF:
-
Time-of-flight
- XEH:
-
Xyloglucan endo-(1,4)-β-d-glucanase
- XET/XTH:
-
Xyloglucan endotransglycosylase/hydrolase
- XG:
-
Xyloglucan
- XG-OS7:
-
Xyloglucan heptasaccharide
- XG-OS8:
-
Xyloglucan octasaccharide
- XG-OS9:
-
Xyloglucan nonasaccharide
- XG-OS:
-
Xyloglucan oligosaccharides
- Xyl:
-
(1,4)-β-d-glucuronoxylan
- Xyl-OS:
-
(1,4)-β-d-glucurono-xylo-oligosaccharides
- WT:
-
Wild-type
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Acknowledgements
This work was supported by the grant No. 2/0058/16 from VEGA, Slovakia to ES, and by the funding from Huaiyin Normal University and the Australian Research Council Linkage Project (DP120100900) to MH. We thank IBH Wilson from the Universität für Bodenkultur, Vienna, Austria, for providing the pPICZα-His/FLAG plasmid, to I Zelko and R Vadkertiova (Institute of Chemistry) for the assistance with fluorescent microscopy, and H Čigašová (Institute of Chemistry) for technical assistance.
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Conceived, designed experiments and analysed data: B.S., Z.F., J.K., S.Š., E.S. and M.H. Z.F. and J.K. determined the primary structure of TmXET6.3, B.S. and E.S. quantified enzyme activities of wild-type and variants, Á.H., B.S. and F.A-M. run electrophoretic analyses, E.S. conducted microscopy analyses, S.Š. constructed variant plasmids and selected hyper-producing clones, D.S. and S.G. worked out activity assays, S.K. built the 3D homology model, BS conducted large-scale bioinformatics analyses and suggested variant sites, V.F. prepared fluorescent oligosaccharides, M.H. conducted phylogeny reconstruction analyses and generated structural graphics. Discussed the data and contributed to writing: B.S., J.K., S.Š., S.K., V.F., E.S. and M.H. E.S. and M.H. designed research and wrote the manuscript.
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Database accession numbers: The nucleotide sequence of TmXET6.3 is available in GenBank under HF968473, the protein sequence of TmXET6.3 in UniprotKB under V5ZEF7, and the structural data of TmXET6.3 in the Protein Model DataBase under PM0081526.
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Stratilová, B., Firáková, Z., Klaudiny, J. et al. Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.). Plant Mol Biol 100, 181–197 (2019). https://doi.org/10.1007/s11103-019-00852-8
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DOI: https://doi.org/10.1007/s11103-019-00852-8