Crystal Structure of Thermostable α-Amylase from Bacillus licheniformis Refined at 1.7 Å Resolution

Kwang Yeon Hwang, Hyun Kyu Song, Changsoo Chang, Jungkyu Lee, Suk Yeong Lee, Kyeong Kyu Kim, Senyon Choe, Robert M. Sweet, Se Won Suh

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

α-Amylases (α-1,4-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 Bacillus 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 Fo>2σFo between 8.0 and 1.7 Å resolution, with root mean square deviations of 0.013 Å 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 is 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 destabilizing 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 responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.

Original languageEnglish
Pages (from-to)251-258
Number of pages8
JournalMolecules and Cells
Volume7
Issue number2
Publication statusPublished - 1997 Apr 30
Externally publishedYes

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Amylases
Enzymes
R388
Catalytic Domain
Ions
Amino Acids
Structural Models
X Ray Crystallography
Bacillus licheniformis
Oligosaccharides
Glycogen
Starch
Bacillus
Zea mays
Chlorides
Hot Temperature
Calcium
Glucose
Peptides
Mutation

ASJC Scopus subject areas

  • Cell Biology
  • Genetics
  • Molecular Biology

Cite this

Crystal Structure of Thermostable α-Amylase from Bacillus licheniformis Refined at 1.7 Å Resolution. / Hwang, Kwang Yeon; Song, Hyun Kyu; Chang, Changsoo; Lee, Jungkyu; Lee, Suk Yeong; Kim, Kyeong Kyu; Choe, Senyon; Sweet, Robert M.; Suh, Se Won.

In: Molecules and Cells, Vol. 7, No. 2, 30.04.1997, p. 251-258.

Research output: Contribution to journalArticle

Hwang, KY, Song, HK, Chang, C, Lee, J, Lee, SY, Kim, KK, Choe, S, Sweet, RM & Suh, SW 1997, 'Crystal Structure of Thermostable α-Amylase from Bacillus licheniformis Refined at 1.7 Å Resolution', Molecules and Cells, vol. 7, no. 2, pp. 251-258.
Hwang, Kwang Yeon ; Song, Hyun Kyu ; Chang, Changsoo ; Lee, Jungkyu ; Lee, Suk Yeong ; Kim, Kyeong Kyu ; Choe, Senyon ; Sweet, Robert M. ; Suh, Se Won. / Crystal Structure of Thermostable α-Amylase from Bacillus licheniformis Refined at 1.7 Å Resolution. In: Molecules and Cells. 1997 ; Vol. 7, No. 2. pp. 251-258.
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abstract = "α-Amylases (α-1,4-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 Bacillus 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 Fo>2σFo between 8.0 and 1.7 {\AA} resolution, with root mean square deviations of 0.013 {\AA} 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 is 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 destabilizing 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 responsible 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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T1 - Crystal Structure of Thermostable α-Amylase from Bacillus licheniformis Refined at 1.7 Å Resolution

AU - Hwang, Kwang Yeon

AU - Song, Hyun Kyu

AU - Chang, Changsoo

AU - Lee, Jungkyu

AU - Lee, Suk Yeong

AU - Kim, Kyeong Kyu

AU - Choe, Senyon

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AU - Suh, Se Won

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N2 - α-Amylases (α-1,4-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 Bacillus 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 Fo>2σFo between 8.0 and 1.7 Å resolution, with root mean square deviations of 0.013 Å 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 is 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 destabilizing 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 responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.

AB - α-Amylases (α-1,4-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 Bacillus 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 Fo>2σFo between 8.0 and 1.7 Å resolution, with root mean square deviations of 0.013 Å 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 is 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 destabilizing 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 responsible 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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