CRYSTAL-STRUCTURE OF A CATALYTIC-SITE MUTANT ALPHA-AMYLASE FROM BACILLUS-SUBTILIS COMPLEXED WITH MALTOPENTAOSE

The X-ray crystal structure of a catalytic-site mutant EQ208 [Glu208 - -> Gln] of alpha-amylase from Bacillus subtilis cocrystallized with ma ltopentaose (G5) and acarbose has been determined by multiple isomorph ous replacement at 2.5 Angstrom resolution. Restrained crystallographi c refinement has resulted in an R-factor of 19.8% in the 7.0 to 2.5 An gstrom resolution range. EQ208 consists of three domains containing a (beta/alpha)(8)-barrel as observed in other a-amylases. Clear connecte d density corresponding to a pentasaccharide was observed, which was c onsidered as the G5 molecule based on the high affinity of EQ208 for G 5 that could replace prebound acarbose or a possible transglycosylatio n product of acarbose. The conformation around the third alpha-(1,4)-g lucosidic 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 cl eft. The amide nitrogen of Gln208 forms a hydrogen bond with the gluco sidic oxygen in the scissile bond between Glc3 and Glc4 (Glc1 is the n on-reducing end glucose residue of the substrate). This hydrogen-bondi ng manner may correspond to that of the protonated state of Glu208 in the initial kinetic complex between wildtype 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 precise ly to the place of the catalytic attack. The carboxyl oxygen atoms of the other catalytic-site residues Asp176 and Asp269 form hydrogen bond s with the oxygen atoms of Glc3. The carboxyl group of Asp176 has non- bonded contacts to the anomeric carbon atom and to the endocyclic oxyg en atom of Glc3. These results suggest that Glu208 acts as a general a cid 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 therm al factor of 13.2 Angstrom(2) among the five sugar residues. Three cal cium ions are found, one of which is positioned near the substrate bin ding site as found in other alpha-amylases and could contribute to sta bilization of the structure of the active site. (C) 1998 Academic Pres s Limited.