Crystal structure of recombinant soybean beta-amylase complexed with beta-cyclodextrin.
In order to study the interaction of soybean beta-amylase with substrate, we solved the crystal structure of beta-cyclodextrin-enzyme complex and compared it with that of alpha-cyclodextrin-enzyme complex. The enzyme was expressed in Escherichia coli at a high level as a soluble and catalytically active protein. The purified recombinant enzyme had properties nearly identical to those of native soybean beta-amylase and formed the same crystals as the native enzyme. The crystal structure of recombinant enzyme complexed with beta-cyclodextrin was refined at 2. 07-A resolution with a final crystallographic R value of 15.8% (Rfree = 21.1%). The root mean square deviation in the position of C-alpha atoms between this recombinant enzyme and the native enzyme was 0.22 A. These results indicate that the expression system established here is suitable for studying structure-function relationships of beta-amylase. The conformation of the bound beta-cyclodextrin takes an ellipsoid shape in contrast to the circular shape of the bound alpha-cyclodextrin. The cyclodextrins shared mainly two glucose binding sites, 3 and 4. The glucose residue 4 was slightly shifted from the maltose binding site. This suggests that the binding site of the cyclodextrins is important for its holding of a cleaved substrate, which enables the multiple attack mechanism of beta-amylase.
Adachi
M
M
Research Institute for Food Science, Kyoto University, Uji Kyoto 611-0011, Japan.
Mikami
B
B
Katsube
T
T
Utsumi
S
S
D001665:Binding Sites; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013025:Glycine max / Q000201:enzymology*; D013329:Structure-Activity Relationship; D001614:beta-Amylase / Q000737:chemistry*; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D010940:Plant Proteins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; D001614:beta-Amylase; D005947:Glucose; C031215:betadex