Molecular characterisation of a xyloglucan oligosaccharide-acting alpha-D-xylosidase from nasturtium (Tropaeolum majus L.) cotyledons that resembles plant 'apoplastic' alpha-D-glucosidases.
We report the isolation, sequencing and analysis of the cDNA corresponding to an alpha-D-xylosidase involved in the mobilisation of xyloglucan from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds. The translated open reading frame (2,808 bp including the stop codon), gave a polypeptide of 935 amino acids. It included the sequences of eleven peptides obtained by endo-proteinase digestion of the protein, and a putative hydrophobic signal sequence characteristic of a protein that is directed through the plasma membrane. The deduced molecular weight of the translated protein was appreciably higher than the molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, suggesting post-translational modification. The protein sequence showed high homology (76.0% identity over 896 amino acids) with a putative alpha-xylosidase sequence from Arabidopsis thaliana and there was homology with several alpha-glucosidases, notably those associated with the plant cell apoplast. The enzyme is a member of Family 31 of the glycosyl hydrolases and it fits into Clade 1 of the phylogenic analysis of alpha-glucosidases. Although in vivo the nasturtium enzyme catalyses mobilisation of cell wall xyloglucan, the homology of its primary sequence with alpha-glucosidases prompted study of its action on a range of alpha-glucosides. It was active against several alpha-(1-->4)-and alpha-(1-->6)-linked substrates, the former being hydrolysed faster. The functional and evolutionary relationships between this alpha-D-xylosidase and plant "apoplastic" alpha-D-glucosidases are discussed.
Crombie
Hazel J
HJ
Department of Biological Sciences, University of Stirling, UK.
Chengappa
Sumant
S
Jarman
Carl
C
Sidebottom
Chris
C
Reid
J S Grant
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D002473:Cell Wall / Q000378:metabolism; D018548:Cotyledon / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans; D019684:Magnoliopsida / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014990:Xylans; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D005936:Glucans; D009844:Oligosaccharides; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D014995:Xylosidases; C047831:alpha-D-xylosidase; D000520:alpha-Glucosidases