IngentaConnect Val216 decides the substrate specificity of -glucosidase in Sacch...

Authors: Yamamoto, Keizo¹; Nakayama, Akifumi²; Yamamoto, Yuka; Tabata, Shiro¹

Source: FEBS Journal, Volume 271, Number 16, August 2004 , pp. 3414-3420(7)

Abstract:

Differences in the substrate specificity of α-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 α-1,4- and α-1,6-glucosidic linkages, two α-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 72.1% sequence identity with 165 amino acid alterations between the two α-glucosidases. These two α-glucosidase genes were subcloned into the pKP1500 expression vector and expressed in Escherichia coli. The purified α-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 consensus region II, which is one of the four regions conserved in family 13 (α-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 α-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 α-1,4- and 1,6-glucosidic linkages of substrates.

Keywords: family 13; α-glucosidase; Saccharomyces cerevisiae; site-directed mutagenesis; substrate specificity

Document Type: Research article

DOI: 10.1111/j.1432-1033.2004.04276.x

Affiliations: 1: Department of Chemistry, Nara Medical University, Japan 2: Nara Prefectural Institute for Hygiene and Environment, Japan

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Val216 decides the substrate specificity of α-glucosidase in Saccharomyces cerevisiae