Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremulaxtremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris.
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.
Kallas
Asa M
AM
Department of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, SE-10691 Stockholm, Sweden.
Piens
Kathleen
K
Denman
Stuart E
SE
Henriksson
Hongbin
H
Fäldt
Jenny
J
Johansson
Patrik
P
Brumer
Harry
H
Teeri
Tuula T
TT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004795:Enzyme Stability; D015870:Gene Expression / Q000502:physiology; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000378:metabolism*; D032107:Populus / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D020033:Protein Isoforms; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D020033:Protein Isoforms; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase