CRYSTAL-STRUCTURE OF THERMOSTABLE ALPHA-AMYLASE FROM BACILLUS-LICHENIFORMIS REFINED AT 1.7 ANGSTROM RESOLUTION

alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) cata lyze the cleavage of alpha-1, 4-glucosidic linkages of starch componen ts, glycogen, and various oligosaccharides. Thermostable alpha-amylase s from Bacillus species are of great industrial importance in the prod uction of corn syrup or dextrose. Thermostable alpha-amylase from Baci llus licheniformis, a monomeric enzyme with molecular mass of 55,200 D a (483 amino acid residues), shows a remarkable heat stability. This e nzyme provides an attractive model for investigating the structural ba sis for thermostability of proteins. The three-dimensional structure o f thermostable alpha-amylase from Bacillus licheniformis has been dete rmined by the multiple isomorphous replacement method of X-ray crystal lography. The structure has been refined to a crystallographic R-facto r of 19.9% for 58,601 independent reflections with F-o>2 sigma Fo betw een 8.0 and 1.7 Angstrom resolution, with root mean square deviations of 0.013 Angstrom from ideal bond lengths and 1.72 degrees from ideal bond angles. The final model consists of 469 amino acid residues and 2 94 water molecules, Missing from the model are the N- and C-termini an d the segment between Trp182 and Asn192. Like other alpha-amylases, th e polypeptide chain folds into three distinct domains. The first domai n (domain A), consisting of 291 residues (from residue 3 to 103 and 20 7 to 396), forms a (beta/alpha)(s)-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between th e third beta-strand and the third alpha-helix of domain A. The third C -terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel. Neither calcium ion n or chloride ion is located near the active site. This study reveals th e architecture of the thermostable alpha-amylase from Bacillus licheni formis. By homology with other alpha-amylases, important active site r esidues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (beta/alpha)(s)-barrel. Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appea rs to be important for thermostability. The factors responsible for th e remarkable thermostability of this enzyme may be increased ionic int eractions, reduced surface area, and increased packing interactions in the interior.