Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.

Article abstract of DOI:10.1271/bbb.65.2058

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 alpha-glucosidase, Aspergillus oryzae alpha-amylase and pig pancreatic alpha-amylase which act on alpha-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 alpha-amylase and pig pancreatic alpha-amylase. A single mutation of Asp199-->Asn, Glu255-->Gln, or Asp329-->Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of alpha-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of alpha-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (alpha-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 k0/Km of that for the wild-type enzyme. Since mutants of other alpha-amylases acting on alpha-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 alpha-1,6-glucosidic bond linkage.

Full text of DOI:10.1271/bbb.65.2058

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Identification of Catalytic and Substrate-binding Site
Residues in Bacillus cereus ATCC7064 Oligo-1,6-
glucosidase
a
a
a
Kunihiko WATANABE , Kazue MIYAKE & Yuzuru SUZUKI
a
Department of Applied Biochemistry, Kyoto Prefectural University
Published online: 22 May 2014.
To cite this article: Kunihiko WATANABE, Kazue MIYAKE & Yuzuru SUZUKI (2001) Identification of Catalytic and Substrate-
binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase, Bioscience, Biotechnology, and Biochemistry,
65:9, 2058-2064, DOI: 10.1271/bbb.65.2058
To link to this article: http://dx.doi.org/10.1271/bbb.65.2058
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