Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.
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.
Department of Chemistry, Structural Biology Laboratory, University of York, Heslington, UK.
A M
AM
Brzozowski
D M
DM
Lawson
J P
JP
Turkenburg
H
H
Bisgaard-Frantzen
A
A
Svendsen
T V
TV
Borchert
Z
Z
Dauter
K S
KS
Wilson
G J
GJ
Davies
D020909:Acarbose / Q000737:chemistry; D001407:Bacillus / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002021:Buffers; D002118:Calcium / Q000737:chemistry; D002240:Carbohydrate Sequence; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry; D005798:Genes, Bacterial; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D014312:Trisaccharides / Q000737:chemistry; D014325:Tromethamine; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D002021:Buffers; D004791:Enzyme Inhibitors; D008024:Ligands; D046911:Macromolecular Substances; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D014312:Trisaccharides; D014325:Tromethamine; C008317:maltotriose; D000516:alpha-Amylases; D002118:Calcium; D020909:Acarbose