Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1764, Issue 4, Pages 688-698Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2006.01.012
Keywords
dextran glucosidase; site-directed mutagenesis; substrate specificity; alpha-amylase; alpha-glucosidase; glycoside hydrolase family 13
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Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta -> alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar k(cat)/k(m) value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta -> alpha loop 4 of B. subtilis oligo- 1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta ->alpha loop 4 were important for high activity to long-chain substrates. (c) 2006 Elsevier B.V. All rights reserved.
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