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Journal of Biochemistry Advance Access originally published online on May 24, 2007
Journal of Biochemistry 2007 142(1):87-93; doi:10.1093/jb/mvm110
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© 2007 The Japanese Biochemical Society.

Molecular Determinants of Substrate Recognition in Thermostable {alpha}-glucosidases Belonging to Glycoside Hydrolase Family 13

Yoshiyuki Tsujimoto*, Hiroyuki Tanaka, Reiko Takemura, Tomohiko Yokogawa, Atsushi Shimonaka, Hiroshi Matsui, Shin-ichi Kashiwabara, Kunihiko Watanabe and Yuzuru Suzuki

Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan

*To whom correspondence should be addressed. Tel: +81-75-703-5669, Fax: +81-75-703-5669, E-mail: yoshi_t{at}kpu.ac.jp

Received March 6, 2007; Accepted May 2, 2007


  Abstract

Bacillus stearothermophilus {alpha}-1,4-glucosidase (BS) is highly specific for {alpha}-1,4-glucosidic bonds of maltose, maltooligosaccharides and {alpha}-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse {alpha}-1,6 bonds of isomaltose, isomaltooligosaccharides and {alpha}-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 ->Ala in region II and Pro258 ->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k0/Km (s–1· mM–1) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the {alpha}-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.

Key Words: {alpha}-glucosidase, glycoside hydrolase family 13, substrate specificity

Abbreviations: BS, Bacillus stearothermophilus {alpha}-1,4-glucosidase; BT, Bacillus thermoglucosidasius oligo-1,6-glucosiade; GH, glycoside hydrolase family; LA, LB medium supplemented with 100 µg/ml ampicillin; pNPG, p-nitro-phenyl-{alpha}-D-glucopyranoside; PAGE, polyacrylamide gel electrophoresis; SAM, Bacillus sp. SAM1606 {alpha}-glucosidase


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