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J. Biochem, 2003, Vol. 133, No. 4 467-474
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Crystal Structures of ß-Amylase from Bacillus cereus var. mycoides in Complexes with Substrate Analogs and Affinity-Labeling Reagents

Takuji Oyama+,1, Hideo Miyake1, Masami Kusunoki2 and Yasunori Nitta§,1

1 Laboratory of Enzyme Chemistry, Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, Osaka 599-8531, and 2 Institute for Protein Research, Osaka University, Suita, Osaka 565-0871

The crystal structures of ß-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 Å 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 ß-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.

+ Present address: Department of Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874.

§ To whom correspondence should be addressed. Tel: +81-72-254-9473, Fax: +81-072-254-9474, E-mail: nitta{at}biochem.osakafu-u.ac.jp


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