Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes alpha-1,4 and alpha-1,6 linkages in polysaccharides at different active sites.
An amylopullulanase from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S. (1995) Biochim. Biophys. Acta 1243, 315-324). Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites. The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids. The molecular mass of the extracellular mature enzyme (Glu33 through Leu1938) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp. KSM-1378. The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids. Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity. The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a "castanet-like" or "bent dumbbell-like" molecule with a diameter of approximately 25 nm.
Hatada
Y
Y
Tochigi Research Laboratories of the Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan.
Igarashi
K
K
Ozaki
K
K
Ara
K
K
Hitomi
J
J
Kobayashi
T
T
Kawai
S
S
Watabe
T
T
Ito
S
S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005936:Glucans; D011134:Polysaccharides; C009109:pullulan; D006026:Glycoside Hydrolases; C082507:amylopullulanase