Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents.
The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.
Oyama
Takuji
T
Laboratory of Enzyme Chemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, Osaka 599-8531. nitta@biochem.osakafu-u.ac.jp
Miyake
Hideo
H
Kusunoki
Masami
M
Nitta
Yasunori
Y
D000345:Affinity Labels; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005936:Glucans / Q000737:chemistry; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism; D001614:beta-Amylase / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D005936:Glucans; D009844:Oligosaccharides; D008320:Maltose; D001614:beta-Amylase; D000520:alpha-Glucosidases; D005947:Glucose