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Journal of Biochemistry Advance Access published online on May 24, 2007
Journal of Biochemistry, 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

Corresponding author: Yoshiyuki Tsujimoto. Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan. TEL: +81-92-642-2584, 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-16-glucosidase (BT) can specifically hydrolyze 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 analyzed 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.


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