Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: isolation and characterization of the major form.
Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cello-oligosaccharides but also to oligosaccharides derived from beta-(1,4)-d-glucuronoxylan, beta-(1,6)-d-glucan, mixed-linkage beta-(1,3; 1,4)-d-glucan and at a relatively low rate also to beta-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage beta-(1,3; 1,4)-d-gluco-oligosaccharides 2.06%, with beta-(1,4)-d-glucuronoxylo-oligosaccharides 0.31% and with beta-(1,6)-d-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.
Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84538 Bratislava, Slovakia.
Eva
E
Stratilová
Fairouz
F
Ait-Mohand
Pavel
P
Rehulka
Sona
S
Garajová
Dana
D
Flodrová
Helena
H
Rehulková
Vladimír
V
Farkas
D000595:Amino Acid Sequence; D002482:Cellulose / Q000378:metabolism; D018525:Germination; D005936:Glucans / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms; D012639:Seeds / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D032388:Tropaeolum / Q000201:enzymology*; D014990:Xylans / Q000378:metabolism*; D047071:beta-Glucans / Q000378:metabolism
D005936:Glucans; D005960:Glucosides; D009844:Oligosaccharides; D010940:Plant Proteins; D020033:Protein Isoforms; D014990:Xylans; D047071:beta-Glucans; C029353:xyloglucan; D002482:Cellulose; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase