1E40

Tris/maltotriose complex of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 2.2A

Summary for 1E40

• Entry DOI: 10.2210/pdb1e40/pdb
• Related: 1BLI 1BPL 1E3X 1E3Z 1E43 1VJS
• Related PRD ID: PRD_900009
• Descriptor: ALPHA-AMYLASE, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: hydrolase, amylase, family 13, maltotriose, tris, complex
• Biological source: BACILLUS AMYLOLIQUEFACIENS
• Total number of polymer chains: 1
• Total formula weight: 55848.29

Authors

Brzozowski, A.M.,Lawson, D.M.,Turkenburg, J.P.,Bisgaard-Frantzen, H.,Svendsen, A.,Borchert, T.V.,Dauter, Z.,Wilson, K.S.,Davies, G.J. (deposition date: 2000-06-27, release date: 2001-06-21, Last modification date: 2023-12-13)

Primary citation

Brzozowski, A.M.,Lawson, D.M.,Turkenburg, J.P.,Bisgaard-Frantzen, H.,Svendsen, A.,Borchert, T.V.,Dauter, Z.,Wilson, K.S.,Davies, G.J.
Structural Analysis of a Chimeric Bacterial Alpha-Amylase. High Resolution Analysis of Native and Ligand Complexes
Biochemistry, 39:9099-, 2000

PubMed Abstract: 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.

PubMed: 10924103
DOI: 10.1021/BI0000317

Experimental method

X-RAY DIFFRACTION (2.2 Å)