Identification of catalytic and substrate-binding site residues in Bacillus cereus ATCC7064 oligo-1,6-glucosidase.
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.
Watanabe
K
K
Department of Applied Biochemistry, Kyoto Prefectural University, Sakyo, Japan. kwatanab@kpu.ac.jp
Miyake
K
K
Suzuki
Y
Y
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D015180:Electrophoresis, Gel, Two-Dimensional; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009834:Oligo-1,6-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation
D009834:Oligo-1,6-Glucosidase