Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and α‐amylases Site‐directed mutagenesis of the conserved two Asp and one Glu residues

FEBS Letters

Kuwano, Kayoko¹  Haga, Keiko¹  Kimura, Kenji²  Yamane, Kunio¹  Ogawa, Shigeyuki¹  Nakamura, Akira¹ 

[1] Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan;Central Laboratory, Oji-Cornstarch Co., Ichihara, Chiba 290, Japan

关键词: Active site;    Amylase;    Catalytic center;    Cyclodextrin glucanotransferase;    Site-directed mutagenesis;    BSA;    bovine serum albumin;    CGTase;    cyclodextrin glucanotransferase;    kb;    kilobase pairs;    PAGE;    polyacrylamide gel electrophoresis;    SDS;    sodium dodecyl sulfate;   

DOI  :  10.1016/0014-5793(92)80398-Z

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of α-amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in α-amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site-directed mutagenesis was carried out. The three mutant CGTases, in which Asp²²⁹, Glu²⁵⁷ and Asp³²⁸ were individually replaced by Asn or Gln, completely lost both their starch-degrading and β-cyclodextrin-forming activities, whereas another mutant CGTase, in which Glu²⁶⁴ replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp²²⁹, Glu²⁵⁷ and Asp³²⁸ play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and α-amylases.

【 授权许可】

Unknown