Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1, 6-glucosidase(Biochemistry & Molecular Biology)
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概要
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Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1, 6-glucosidase (EC 3. 2. 1. 10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1, 6-glucosidase with those of Saccharomyces carlsbergensis α-glucosidase, Aspergillus oryzae α-amylase and pig pancreatic α-amylase which act on α-1, 4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1, 6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae α-amylase and pig pancreatic α-amylase. A single mutation of Aspl99→Asn, Glu255→Gln, or Asp329→Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of α-1, 6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1, 6-glucosidase for the hydrolysis of α-1, 6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (α-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328→Asn caused the essential loss in activity, while the mutation His103→Asn yielded a mutant enzyme that retained 59% of the k_0/K_m of that for the wild-type enzyme. Since mutants of other α-amylases acting on α-1, 4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by His103→Asn mutation in B. cereus oligo-1, 6-glucosidase revealed the distinguished role of His103 for the hydrolysis of α-1, 6-glucosidic bond linkage.
- 社団法人日本農芸化学会の論文
- 2001-09-23
著者
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Watanabe Kunihiko
Department Of Chemical Industry Science University Of Tokyo
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Suzuki Yuzuru
Department Of Applied Biochemistry Kyoto Prefectural University
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MIYAKE Kazue
Department of Applied Biochemistry, Kyoto Prefectural University
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Miyake Kazue
Department Of Applied Biochemistry Kyoto Prefectural University
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Watanabe Kunihiko
Department Of Applied Biochemistry Kyoto Prefectural University
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Suzuki Yuzuru
Department Of Agricultural Chemistry Kyoto Prefectural University
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