title: Differences in the thermostabilities of barley (1----3,1----4)-beta-glucanases are only partly determined by N-glycosylation.
abstract: Barley (1----3,1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzyme EII carries 4% by weight carbohydrate and is more stable at elevated temperatures than isoenzyme EI, which has no associated carbohydrate. The relationship between carbohydrate content and thermostability has been investigated by treatment of the two isoenzymes with N-glycopeptidase F (EC 3.5.1.52). Removal of carbohydrate from isoenzyme EII results in a decrease in the enzyme's thermostability, but treatment of isoenzyme EI with the N-glycopeptidase F has no effect. In addition, removal of a single N-glycosylation site in isoenzyme EII (Asn190-Ala-Ser) by site-directed mutagenesis of the corresponding cDNA led to a reduction in thermostability, while the introduction of this site into isoenzyme EI enhanced stability. We conclude that N-glycosylation of Asn190 enhances the stability of isoenzyme EII at elevated temperatures, but that other factors related to their primary structures also contribute to the differences in thermostabilities of the barley (1----3,1----4)-beta-glucanases.
authors.affiliation: Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
authors.forename: D N
authors.initials: DN
authors.lastname: Doan
authors.forename: G B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D001467:Hordeum / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D004247:DNA; D006026:Glycoside Hydrolases; C028020:licheninase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase