Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13.
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 k(0)/K(m) (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.
Tsujimoto
Yoshiyuki
Y
Department of Applied Biochemistry, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto 606-8522, Japan. yoshi-t@kpu.ac.jp
Tanaka
Hiroyuki
H
Takemura
Reiko
R
Yokogawa
Tomohiko
T
Shimonaka
Atsushi
A
Matsui
Hiroshi
H
Kashiwabara
Shin-ichi
S
Watanabe
Kunihiko
K
Suzuki
Yuzuru
Y
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000145:classification / Q000201:enzymology; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000145:classification; D006868:Hydrolysis; D007534:Isomaltose / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D007534:Isomaltose; D008320:Maltose; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases