tailieunhanh - Báo cáo khoa học: Val216 decides the substrate specificity of a-glucosidase in Saccharomyces cerevisiae

Differences in the substrate specificity ofa-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzinga-1,4- and a-1,6-glu-cosidic linkages, twoa-glucosidases,maltase and isomaltase, from Saccharomyces cerevisiaewere cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues, respectively. There was sequence identity with 165 amino acid alterations between the two a-glucosidases. . | Eur. J. Biochem. 271 3414-3420 2004 FEBS 2004 doi Val216 decides the substrate specificity of a-glucosidase in Saccharomyces cerevisiae Keizo Yamamoto1 Akifumi Nakayama2 Yuka Yamamoto and Shiro Tabata1 1 Department of Chemistry Nara Medical University Japan 2Nara Prefectural Institute for Hygiene and Environment Japan Differences in the substrate specificity of a-glucosidases should be due to the differences in the substrate binding and the catalytic domains of the enzymes. To elucidate such differences of enzymes hydrolyzing a-1 4- and a-1 6-glu-cosidic linkages two a-glucosidases maltase and isomaltase from Saccharomyces cerevisiae were cloned and analyzed. The cloned yeast isomaltase and maltase consisted of 589 and 584 amino acid residues respectively. There was sequence identity with 165 amino acid alterations between the two a-glucosidases. These two a-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified a-glucosidases showed the same substrate specificities as those of their parent native glucosidases. Chimeric enzymes constructed from isomaltase by exchanging with maltase fragments were characterized by their substrate specificities. When the con sensus region II which is one of the four regions conserved in family 13 a-amylase family is replaced with the maltase type the chimeric enzymes alter to hydrolyze maltose. Three amino acid residues in consensus region II were different in the two a-glucosidases. Thus we modified Val216 Gly217 and Ser218 of isomaltase to the maltase-type amino acids by site-directed mutagenesis. The Val216 mutant was altered to hydrolyze both maltose and isomaltose but neither the Gly217 nor the Ser218 mutant changed their substrate specificity indicating that Val216 is an important residue discriminating the a-1 4- and 1 6-glucosidic linkages of substrates. Keywords family 13 a-glucosidase Saccharomyces cere-visiae site-directed .

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