Source:http://linkedlifedata.com/resource/pubmed/id/10924103
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
31
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| pubmed:dateCreated |
2000-9-7
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| pubmed:databankReference | |
| pubmed:abstractText |
Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 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 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis 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 attributed 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 Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. 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-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Acarbose,
http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Buffers,
http://linkedlifedata.com/resource/pubmed/chemical/Calcium,
http://linkedlifedata.com/resource/pubmed/chemical/Enzyme Inhibitors,
http://linkedlifedata.com/resource/pubmed/chemical/Ligands,
http://linkedlifedata.com/resource/pubmed/chemical/Macromolecular Substances,
http://linkedlifedata.com/resource/pubmed/chemical/Oligosaccharides,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Fusion Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Trisaccharides,
http://linkedlifedata.com/resource/pubmed/chemical/Tromethamine,
http://linkedlifedata.com/resource/pubmed/chemical/alpha-Amylases,
http://linkedlifedata.com/resource/pubmed/chemical/maltotriose
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| pubmed:status |
MEDLINE
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| pubmed:month |
Aug
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| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
8
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| pubmed:volume |
39
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
9099-107
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| pubmed:dateRevised |
2008-11-21
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| pubmed:meshHeading |
pubmed-meshheading:10924103-Acarbose,
pubmed-meshheading:10924103-Bacillus,
pubmed-meshheading:10924103-Bacterial Proteins,
pubmed-meshheading:10924103-Binding Sites,
pubmed-meshheading:10924103-Buffers,
pubmed-meshheading:10924103-Calcium,
pubmed-meshheading:10924103-Carbohydrate Sequence,
pubmed-meshheading:10924103-Computer Simulation,
pubmed-meshheading:10924103-Crystallization,
pubmed-meshheading:10924103-Crystallography, X-Ray,
pubmed-meshheading:10924103-Enzyme Inhibitors,
pubmed-meshheading:10924103-Genes, Bacterial,
pubmed-meshheading:10924103-Ligands,
pubmed-meshheading:10924103-Macromolecular Substances,
pubmed-meshheading:10924103-Models, Molecular,
pubmed-meshheading:10924103-Molecular Sequence Data,
pubmed-meshheading:10924103-Oligosaccharides,
pubmed-meshheading:10924103-Recombinant Fusion Proteins,
pubmed-meshheading:10924103-Trisaccharides,
pubmed-meshheading:10924103-Tromethamine,
pubmed-meshheading:10924103-alpha-Amylases
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| pubmed:year |
2000
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| pubmed:articleTitle |
Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.
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| pubmed:affiliation |
Department of Chemistry, Structural Biology Laboratory, University of York, Heslington, UK.
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| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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