Structural analysis of a chimeric bacterial alpha-amylase. High-resolutionanalysis of native and ligand complexes

Several chimeric alpha-amylases genes were constructed by an in vivo recomb ination technique from the Bacillus amyloliquefaciens and Bacillus lichenif ormis genes. One of the fusion amylases (hereafter BA2), consisting of resi dues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2 .2 and 1.7 Angstrom. The 3-dimensional structure of the native enzyme was s olved by multiple isomorphous replacement, and refined at a resolution of 1 .7 Angstrom. It consists of 483 amino acids, organized similarly to the kno wn B, lichiniformis alpha alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attribut ed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca2+ bindi ng site of fungal alpha-amylases. The third calcium ion is found at the int erface of the A and C domains. BA2 contains a fourth calcium site, not obse rved in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta beta-sheets. Three acid residues (Glu2 61, Asp328, and Asp231) form an active site similar to that seen in other a mylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-c enter carboxylates. Kinetic data reveal that BA2 displays properties interm ediate to those of its parents. Data for crystals soaked in maltooligosacch arides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously describe d for any alpha-amylase structure, the biological function of which is uncl ear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 Angstrom, reveal a decasaccharide moiety, spanning the -7 to +3 sub sites of the enzyme. The unambiguous presence of three unsaturated rings in the H-2(3) half-chair/E-2 envelope conformation, adjacent to three 6-deoxy pyranose units, clearly demonstrates synthesis of this acarbose-derived dec asaccharide by a two-step transglycosylation mechanism.