Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose

Abstract

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.