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KMID : 0578319970070020251
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Molecules and Cells
1997 Volume.7 No. 2 p.251 ~ p.258
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Crystal structure of Thermostable α-amylase from Bacillus licheniformis refined at 1.7 A Resolution
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Hwang, Kwang Yeon
Song, Hyun Kyu/Changm Changsoo/Lee, Hungkyu/Lee, Suk Yeong/Kim, Kyeong Kyu/Choe, Senyon
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Abstract
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α-Amylases (α-2,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of α-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides. Thermostable α-amylases from Bacillu species are of great industrial importance in the production of corn syrup or dextrose. Thermostable α-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable α-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F_(0)>2σ_(?0) between 8.0 and 1.7Å resolution, with root mean square deviations of 0.013Å from ideal bond lengths and 1.72˚ from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other α-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (β/α)_(8)-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third β-strand and the third α-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel β-barrel. Neither calcium ion nor chloride ion located near the active site. This study reveals the architecture of the thermostable α-amylase from Bacillus licheniformis. By homology with other α-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (β/α)_(8)-barrel. Since many of the stabilizing and destablilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability. The factors reponsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.
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