Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose
The X-ray crystal structure of a catalytic-site mutant EQ208 [Glu208 → Gln] of α-amylase from Bacillus subtilis cocrystallized with maltopentaose (G5) and acarbose has been determined by multiple isomorphous replacement at 2.5 Å resolution. Restrained crystallographic refinement has resulted in an R...
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Published in | Journal of molecular biology Vol. 277; no. 2; pp. 393 - 407 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Elsevier Ltd
27.03.1998
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Subjects | |
Online Access | Get full text |
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Summary: | The X-ray crystal structure of a catalytic-site mutant EQ208 [Glu208 → Gln] of α-amylase from
Bacillus subtilis cocrystallized with maltopentaose (G5) and acarbose has been determined by multiple isomorphous replacement at 2.5 Å resolution. Restrained crystallographic refinement has resulted in an
R-factor of 19.8% in the 7.0 to 2.5 Å resolution range. EQ208 consists of three domains containing a (β/α)
8-barrel as observed in other α-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 α-(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 Å
2 among the five sugar residues. Three calcium ions are found, one of which is positioned near the substrate binding site as found in other α-amylases and could contribute to stabilization of the structure of the active site. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0022-2836 1089-8638 |
DOI: | 10.1006/jmbi.1997.1599 |