Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris

AM Kallas, K Piens, SE Denman, H Henriksson, J Faldt, P Johansson, H Brumer, TT Teeri*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.

Original languageEnglish
Pages (from-to)105-113
Number of pages9
JournalBIOCHEMICAL JOURNAL
Volume390
DOIs
Publication statusPublished - 15 Aug 2005
MoE publication typeA1 Journal article-refereed

Keywords

  • glycoside hydrolase family 16 (GH16)
  • hybrid aspen (Populus tremula x tremuloides)
  • Pichia pastoris
  • protein glycosylation
  • protein MS
  • xyloglucan endotransglycosylase (XET)
  • AZUKI-BEAN EPICOTYLS
  • CELL-WALLS
  • MOLECULAR CHARACTERIZATION
  • COLORIMETRIC ASSAY
  • NASTURTIUM SEEDS
  • GLYCOSYLATION
  • ENZYMES
  • PLANTS
  • OLIGOSACCHARIDES
  • PURIFICATION

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