Structure of Bacillus amyloliquefaciens alpha-amylase at high resolution: implications for thermal stability

Abstract

The crystal structure of Bacillus amyloliquefaciens alpha-amylase (BAA) at 1.4 A resolution revealed ambiguities in the thermal adaptation of homologous proteins in this family. The final model of BAA is composed of two molecules in a back-to-back orientation, which is likely to be a consequence of crystal packing. Despite a high degree of identity, comparison of the structure of BAA with those of other liquefying-type alpha-amylases indicated moderate discrepancies at the secondary-structural level. Moreover, a domain-displacement survey using anisotropic B-factor and domain-motion analyses implied a significant contribution of domain B to the total flexibility of BAA, while visual inspection of the structure superimposed with that of B. licheniformis alpha-amylase (BLA) indicated higher flexibility of the latter in the central domain A. Therefore, it is suggested that domain B may play an important role in liquefying alpha-amylases, as its rigidity offers a substantial improvement in thermostability in BLA compared with BAA.

Figure 1

Superimposition of BAA (yellow) and BLA (red) structures; the thickness of the coils shows their corresponding isotropic B factors. The ellipses indicate the three distinct domains in amylases named A, B and C. (a) The final model composed of two monomers (chain A, left; chain B, right) in a back-to-back orientation with respect to each other. (b) Interaction between chains A and B in the dimeric form through hydrogen bonding of Asn222 from chain A to the nitrogen and carbonyl group of chain B; calcium and sodium ions are presented as red and yellow spheres, respectively. Distances are specified in Å.

Figure 2

Sequence alignment of BAA, BA2, BLA and BStA calculated with the program ClustalW. Secondary structures are presented above the alignment. α-Helices are represented by α, β-strands by β and 3₁₀ helices by η. Conserved residues are specified by a red background and similar residues that belong to the same family are shown as red letters.

Figure 3

Difference Fourier electron-density map contoured at 2.5σ at metal-binding sites after refinement of the entire model excluding the metal ions. The assigned densities for ions are labelled. (a) Ca–Na–Ca metal-binding site. (b) Third calcium-binding site at the interface of domains C and A. (c) Fourth calcium-binding site in domain C.

Figure 4

Superposition of BAA (green) and BLA (red). (a) The bulge created by two extra residues in BAA and main-chain deviation from the critical Gly178. (b) Superimposition of the BAA (yellow) and BLA (red) structures; the thickness of the coils represents their isotropic B factors in the TSD3 region. Calcium and sodium ions are presented as cyan and magenta spheres, respectively.

Figure 5

The TLSMD optimization algorithm matrix for BAA as sequential segments. The main-chain anisotropic ADPs for the region of residues 177–185, a loop located at the surface of domain B, differs considerably from the rest, implying greater internal flexibility. Loops with greater flexibility are shaded in grey.

Figure 6

The cis-peptide bond stabilized by the interaction of Glu186 with Lys277 and Asn273. (a) The cis-peptide bond is shown by an arrow. (b) Differences in the side-chain conformation and hydrogen-bonding features of Asn in BAA (green) and Asp in the same position in BLA (cyan). Distances are specified in Å.