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Alpha-amylase PDB id
Protein chain
425 a.a.
_CA ×3
Waters ×146

References listed in PDB file
Key reference
Title Crystal structure of a catalytic-Site mutant alpha-Amylase from bacillus subtilis complexed with maltopentaose.
Authors Z.Fujimoto, K.Takase, N.Doui, M.Momma, T.Matsumoto, H.Mizuno.
Ref. J Mol Biol, 1998, 277, 393-407. [DOI no: 10.1006/jmbi.1997.1599]
PubMed id 9514750
The X-ray crystal structure of a catalytic-site mutant EQ208 [Glu208-->Gln] of alpha-amylase from Bacillus subtilis cocrystallized with maltopentaose (G5) and acarbose has been determined by multiple isomorphous replacement at 2.5 A resolution. Restrained crystallographic refinement has resulted in an R-factor of 19.8% in the 7.0 to 2.5 A resolution range. EQ208 consists of three domains containing a (beta/alpha)8-barrel as observed in other alpha-amylases. Clear connected density corresponding to a pentasaccharide was observed, which was considered as the G5 molecule based on the high affinity of EQ208 for G5 that could replace pre-bound acarbose or a possible transglycosylation product of acarbose. The conformation around the third alpha-(1,4)-glucosidic bond makes a sharp turn, allowing the substrate to fit into the L-shaped cleft. Aromatic residues build the walls of the substrate binding cleft and leucine residues form the inner curvature of the cleft. The amide nitrogen of Gln208 forms a hydrogen bond with the glucosidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the non-reducing end glucose residue of the substrate). This hydrogen-bonding manner may correspond to that of the protonated state of Glu208 in the initial kinetic complex between wild-type enzyme and substrate. The amide oxygen of Gln208 is anchored by two hydrogen bonds with Ala177 and a water molecule, assisting to make the amide proton point precisely to the place of the catalytic attack. The carboxyl oxygen atoms of the other catalytic-site residues Asp176 and Asp269 form hydrogen bonds with the oxygen atoms of Glc3. The carboxyl group of Asp176 has non-bonded contacts to the anomeric carbon atom and to the endocyclic oxygen atom of Glc3. These results suggest that Glu208 acts as a general acid and Asp176 as a general base. Glc3 forms seven hydrogen bonds with the surrounding protein groups and a stacking interaction with Tyr62, which is consistent with the fact that Glc3 has the lowest mean thermal factor of 13.2 A2 among the five sugar residues. Three calcium ions are found, one of which is positioned near the substrate binding site as found in other alpha-amylases and could contribute to stabilization of the structure of the active site.
Figure 5.
Figure 5. Stereoview of the G5-binding structure of EQ208. Residues involved in the hydrophilic interaction are colored by elements and those in the hydrophobic interaction are shown in orange.
Figure 6.
Figure 6. Schematic drawing of the hydrogen-bonding network between EQ208 and G5. Two water molecules mediating hydrogen bonds and hydrophobic residues interacting with G5 are also indicated.
The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 277, 393-407) copyright 1998.
Secondary reference #1
Title Crystallization and preliminary X-Ray studies of wild type and catalytic-Site mutant alpha-Amylase from bacillus subtilis.
Authors H.Mizuno, Y.Morimoto, T.Tsukihara, T.Matsumoto, K.Takase.
Ref. J Mol Biol, 1993, 234, 1282-1283.
PubMed id 8263932
Secondary reference #2
Title Site-Directed mutagenesis of active site residues in bacillus subtilis alpha-Amylase.
Authors K.Takase, T.Matsumoto, H.Mizuno, K.Yamane.
Ref. Biochim Biophys Acta, 1992, 1120, 281-288. [DOI no: 10.1016/0167-4838(92)90249-D]
PubMed id 1576155
Full text Abstract
Secondary reference #3
Title Changes in the properties and molecular weights of bacillus subtilis m-Type and n-Type alpha-Amylases resulting from a spontaneous deletion.
Authors K.Yamane, Y.Hirata, T.Furusato, H.Yamazaki, A.Nakayama.
Ref. J Biochem (tokyo), 1984, 96, 1849-1858.
PubMed id 6099357
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