1e3x

File:1e3x.gif

spinqualitylabelsall models 1e3x, resolution 1.9Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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 Known structural/functional Site: . 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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