title: Five crucial carboxyl residues of 1,2-alpha-mannosidase from Aspergillus saitoi (A. phoenicis), a food microorganism, are identified by site-directed mutagenesis.
abstract: An acidic 1,2-alpha-mannosidase from fungus, Aspergillus saitoi (now designated Aspergillus phoenicis), is highly specific for 1,2-alpha-mannosidic linkage in the high-mannose type oligosaccharide at pH 5.0. The predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-alpha-mannosidases from fungi, yeast and mouse. Active site determination of the enzyme expressed in Saccharomyces cerevisiae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Glu and of the Glu-residues, Glu-273, Glu-411, Glu-414 and Glu-474, to Asp altered the drastic decrease of specific activities with Man alpha 1-2Man-OMe and Man9-GlcNAc2-PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably in the ionized COO- form, whereas one of four glutamic acid residues, probably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that three glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probably binding sites of substrate.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Yoshida
authors.forename: E
authors.initials: E
authors.lastname: Ichishima
mesh_terms: D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002384:Catalysis; D005516:Food Microbiology; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase