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

Biochem J. 2005 Aug 15;390(Pt 1):105-13. doi: 10.1042/BJ20041749.

Abstract

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremulaxtremuloides) 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 alpha-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 Asn93 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 (Asn93-->Ser) mutant.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Enzyme Stability
  • Gene Expression / physiology
  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism*
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Mutation
  • Pichia / metabolism*
  • Populus / enzymology*
  • Protein Conformation
  • Protein Denaturation
  • Protein Isoforms
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Temperature

Substances

  • Protein Isoforms
  • Glycosyltransferases
  • xyloglucan endotransglycosylase