title: Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii alpha-L-arabinofuranosidase 54.
abstract: A role for N-linked oligosaccharides on the biochemical properties of recombinant alpha-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn(83)-Thr-Thr and Asn(202)-Ser-Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn(83), Asn(202), and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn(202) may contribute to thermostability and catalysis.
authors.affiliation: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-hiroshima 739-0046, Japan. tkoseki@tds1.tr.yamagata-u.ac.jp
authors.forename: Takuya
authors.initials: T
authors.lastname: Koseki
authors.forename: Yozo
authors.initials: Y
authors.lastname: Miwa
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Mese
authors.forename: Akimasa
authors.initials: A
authors.lastname: Miyanaga
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Wakagi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Shoun
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Matsuzawa
authors.forename: Katsumi
authors.initials: K
authors.lastname: Hashizume
mesh_terms: D001230:Aspergillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D004586:Electrophoresis; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase