Structural Analysis of a Chimeric Bacterial α-Amylase. High-Resolution Analysis of Native and Ligand Complexes^,

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.

Differential Regulation of a Hyperthermophilic α-Amylase with a Novel (Ca,Zn) Two-metal Center by Zinc

“…1). A similar carbohydratebinding site was reported for a chimeric Bacillus amyloliquefaciens/licheniformis ␣-amylase (32). The third acarbose-binding site (Ac-III) is located at the surface of domain B at a distance of ϳ30 Å from the active site (Fig.…”

Tyrosine 105 and Threonine 212 at Outermost Substrate Binding Subsites –6 and +4 Control Substrate Specificity, Oligosaccharide Cleavage Patterns, and Multiple Binding Modes of Barley α-Amylase 1

“…The structures validate modeled substrate complexes and subsite maps (8,12,24,25) by highlighting (i) aromatic stacking and hydrogen bonds between carbohydrate and protein (9,10,21,24,26,27), (ii) conformational features of the bound carbohydrate (8,21,28), (iii) conserved geometry of the catalytic site (10,14,15,21,22,29,30), and (iv) substrate binding motifs in ␤ 3 ␣ loops of the catalytic (␤/␣) 8 barrel (15). The macromolecular substrate starch most probably also interacts with distinct areas outside the cleft as suggested by oligosaccharide occupation at so-called surface or secondary sites in several structures from GH-H (10,14,28,31). This additional substrate binding is only proven, however, for starch binding domains of family 20 (afmb.…”

“…Due to enormous diversity in the binding loops, the ␣-amylase family, also referred to as glycoside hydrolase clan H (GH-H) 1 consisting of glycoside hydrolase families 13 (GH13), 70, and 77 comprises almost 30 specificities (15)(16)(17)(18)(19). Substrate analogs are very rarely seen to fill the entire binding site in crystal structures, one example being a Bacillus licheniformis/Bacillus amyloliquefaciens ␣-amylase chimera accommodating at subsites Ϫ7 through ϩ3 a decasaccharide inhibitor derived by transglycosylation from the pseudotetrasaccharide acarbose (14). Related inhibitors cover the only five subsite long binding crevice in pancreatic ␣-amylase (8,9,20), and occupy part of the longer binding sites in microbial ␣-amylases (11,13,21) and in cyclodextrin glucosyltransferase (CGTase) (16,22,23).…”

Oligosaccharide Binding to Barley α-Amylase 1

“…Structural studies of ␣-amylases in complex with substrate analogues have received much attention in the past decade (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The pseudotetrasaccharide inhibitor acarbose was used for the vast majority of these studies.…”