title: N-linked oligosaccharides of Aspergillus awamori feruloyl esterase are important for thermostability and catalysis.
abstract: A unique N-linked glycosylation motif (Asn(79)-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward alpha-naphthylbutyrate (C4), alpha-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased Km and decreased k(cat) for N79A, and to a considerably decreased k(cat) for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.
authors.affiliation: National Research Institute of Brewing, Japan. tkoseki@tds1.tr.yamagata-u.ac.jp
authors.forename: Takuya
authors.initials: T
authors.lastname: Koseki
authors.forename: Kenji
authors.initials: K
authors.lastname: Takahashi
authors.forename: Takashi
authors.initials: T
authors.lastname: Handa
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Yamane
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.forename: Katsumi
authors.initials: K
authors.lastname: Hashizume
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004795:Enzyme Stability; D004952:Esters / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides; D013696:Temperature
chemical_list: D004952:Esters; D009844:Oligosaccharides; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase