Biochem. J. (2005) 390
(105–113) (Printed in Great Britain)
Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula×tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
Åsa M. KALLAS*, Kathleen PIENS*, Stuart E. DENMAN*1, Hongbin HENRIKSSON*, Jenny FÄLDT*, Patrik JOHANSSON†, Harry BRUMER, III* and Tuula T. TEERI*2
*Department of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, SE-10691 Stockholm, Sweden, and †Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, Box 596, SE-75124 Uppsala, Sweden
The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula×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 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.
Key words: glycoside hydrolase family 16 (GH16), hybrid aspen (Populus tremula×tremuloides), Pichia pastoris, protein glycosylation, protein MS, xyloglucan endotransglycosylase (XET).
Abbreviations used: BCA, disodium 2,2´-bicinchoninate; endo H, endoglycosidase H; ESI, electrospray ionization; EST, expressed sequence tag; GH16, glycoside hydrolase family 16; MaxEnt1, Maximum EntropyTM 1 algorithm; Ptt, Populus tremula×tremuloides; XET, xyloglucan endotransglycosylase; XGO, xyloglucan oligosaccharide; XLLGol, reduced xyloglucan-derived nonasaccharide.
1Present address: CSIRO Livestock Industries, 306 Carmody Road, St. Lucia, Qld 4067, Australia.
2To whom correspondence should be addressed (email tuula@biotech.kth.se).
Received 15 October 2004/16 February 2005; accepted 1 April 2005
Published as BJ Immediate Publication 1 April 2005, doi:10.1042/BJ20041749
The Biochemical Society, London ©2005
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