Molecular characterisation of a xyloglucan oligosaccharide-acting α-D-xylosidase from nasturtium (Tropaeolum majus L.) cotyledons that resembles plant 'apoplastic' α-D-glucosidases

Abstract

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.

Potential Role for Purple Acid Phosphatase in the Dephosphorylation of Wall Proteins in Tobacco Cells

“…Peptides from spot 3 (in the medium) matched the ESTs gi|52834489, gi|76867473, and gi|39866468. Seven peptide fragments detected from these ESTs were 90% identical to nasturtium a-xylosidase (Crombie et al, 2002). Among these seven fragments, four peptides were identical to those from spot 1 (in the walls).…”

“…Peptides from spot 1 matched the ESTs gi|52834489 and gi|39866468. Four peptide sequences detected from these ESTs with P , 0.05 were 93% identical to nasturtium (Tropaeolum majus) a-xylosidase (Crombie et al, 2002). Peptide fragments from spot 1 also matched potato (Solanum tuberosum) a-glucosidase and Arabidopsis a-xylosidase when the NCBInr database was used.…”

Changes in α-Xylosidase during Intact and Auxin-Induced Growth of Pine Hypocotyls

“…Two plant a-xylosidase genes, one from Arabidopsis thaliana (Sampedro et al 2001) and the other from nasturtium (Crombie et al 2002) have been cloned previously. Based on both sequences (Fig.…”

“…They have an apparent molecular mass on SDS-PAGE of 85 kDa for the two first and of 87 kDa for the latter one. Recently, an a-xylosidase (AtXYL1) from Arabidopsis thaliana (Sampedro et al 2001) and another one from Tropaeolum majus (Crombie et al 2002) have been cloned. Both a-xylosidases (GenBank accession nos.…”

Substrate Recognition of Escherichia coli YicI (.ALPHA.-Xylosidase)

“…The enzyme hardly ever hydrolyzes maltose, isomaltose, isomaltotriose, panose or maltotriose. Accordingly, YicI is highly specific for α xylosidic linkages, whereas other reported GH 31 α xylosidases, such as Sulfolobus (XylS), 6) nasturtium 23) and Arabidopsis 24) α xylosidases, show significant α glucosidase activity.…”