Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
2005 (English)In: Biochemical Journal, ISSN 0264-6021, Vol. 390, 105-113 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2005. Vol. 390, 105-113 p.
glycoside hydrolase family 16 (GH16), hybrid aspen (Populus tremula x tremuloides), Pichia pastoris, protein glycosylation, protein MS, xyloglucan endotransglycosylase (XET)
IdentifiersURN: urn:nbn:se:kth:diva-5704DOI: 10.1042/BJ20041749ISI: 000231492800012ScopusID: 2-s2.0-23944474570OAI: oai:DiVA.org:kth-5704DiVA: diva2:10155
QC 201009032006-05-112006-05-112010-09-03Bibliographically approved