1e3x
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NATIVE STRUCTURE OF CHIMAERIC AMYLASE FROM B. AMYLOLIQUEFACIENS AND B. LICHENIFORMIS AT 1.92A
OverviewOverview
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.
About this StructureAbout this Structure
1E3X is a Single protein structure of sequence from Bacillus amyloliquefaciens with CA and NA as ligands. Active as Alpha-amylase, with EC number 3.2.1.1 Structure known Active Site: ACT. Full crystallographic information is available from OCA.
ReferenceReference
Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes., Brzozowski AM, Lawson DM, Turkenburg JP, Bisgaard-Frantzen H, Svendsen A, Borchert TV, Dauter Z, Wilson KS, Davies GJ, Biochemistry. 2000 Aug 8;39(31):9099-107. PMID:10924103
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