title: The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase.
abstract: N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5-8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid residues 435-455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Miyazaki
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yashiro
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan tonozuka@cc.tuat.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000187:drug effects*; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013696:Temperature
chemical_list: D001216:Asparagine; D006026:Glycoside Hydrolases; C047371:isopullulanase
keywords: N-glycosylation; crystal structure; glycoprotein; glycoside hydrolase; thermostability