5 CRUCIAL CARBOXYL RESIDUES OF 1,2-ALPHA-MANNOSIDASE FROM ASPERGILLUS-SAITOI (ASPERGILLUS-PHOENICIS), A FOOD MICROORGANISM, ARE IDENTIFIED BY SITE-DIRECTED MUTAGENESIS

An acidic 1,2-alpha-mannosidase from fungus, Aspergillus saitoi (now d esignated Aspergillus phoenicis), is highly specific for 1,2-alpha-man nosidic linkage in the high-mannose type oligosaccharide at pH 5.0. Th e predicted amino acid sequence of several peptide regions, including aspartic acid and glutamic acid, bears striking similarities to 1,2-al pha-mannosidases from fungi, yeast and mouse. Active site determinatio n of the enzyme expressed in Saccharomyces cerevisiae cells was perfor med 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 alt ered the drastic decrease of specific activities with Man alpha 1-2Man -OMe and Man(9)-GlcNAc(2)-PA as substrates and shifted the optimal pH of the mutant enzymes. From the present results, Asp-269 is probably i n the ionized COO- form, whereas one of four glutamic acid residues, p robably Glu-411, is the un-ionized COOH form according to the analogy of a plausible mechanism for lysozyme catalysis. It is assumed that th ree glutamic acid residues, Glu-273, Glu-414, and Glu-474, are probabl y binding sites of substrate. (C) 1997 Academic Press.