The first slowest motion modes of NpAS (A) and DgAS (B) revealed by the ANM.

<p>The length of the cone in each C_α atom represents the magnitude of movement and its direction indicates the moving direction.</p

The contact maps for the native (A), LNNC = 100 (B), 200 (C) and 500 (C) states of DgAS (D).

<p>Each contact is represented by a “+” mark.</p

Insight into the Structure, Dynamics and the Unfolding Property of Amylosucrases: Implications of Rational Engineering on Thermostability

<div><p>Amylosucrase (AS) is a kind of glucosyltransferases (E.C. 2.4.1.4) belonging to the Glycoside Hydrolase (GH) Family 13. In the presence of an activator polymer, in vitro, AS is able to catalyze the synthesis of an amylose-like polysaccharide composed of only α-1,4-linkages using sucrose as the only energy source. Unlike AS, other enzymes responsible for the synthesis of such amylose-like polymers require the addition of expensive nucleotide-activated sugars. These properties make AS an interesting enzyme for industrial applications. In this work, the structures and topology of the two AS were thoroughly investigated for the sake of explaining the reason why <em>Deinococcus geothermalis</em> amylosucrase (DgAS) is more stable than <em>Neisseria polysaccharea</em> amylosucrase (NpAS). Based on our results, there are two main factors that contribute to the superior thermostability of DgAS. On the one hand, DgAS holds some good structural features that may make positive contributions to the thermostability. On the other hand, the contacts among residues of DgAS are thought to be topologically more compact than those of NpAS. Furthermore, the dynamics and unfolding properties of the two AS were also explored by the gauss network model (GNM) and the anisotropic network model (ANM). According to the results of GNM and ANM, we have found that the two AS could exhibit a shear-like motion, which is probably associated with their functions. What is more, with the discovery of the unfolding pathway of the two AS, we can focus on the weak regions, and hence designing more appropriate mutations for the sake of thermostability engineering. Taking the results on structure, dynamics and unfolding properties of the two AS into consideration, we have predicted some novel mutants whose thermostability is possibly elevated, and hopefully these discoveries can be used as guides for our future work on rational design.</p> </div

The cross-correlation maps for the first 40 slow modes of NpAS (A) and DgAS (B).

<p>The A-domain is decomposed into three parts because of the two insertion domains, i.e. B and B′. A1 part is composed of α1–α2 and β1–β3. A2 part is composed of α3–α6 and β4–β7. A3 part is composed of α7–α8 and β8.</p

The Number of Contact per Residue (NCPR) for each domain of NpAS and DgAS.

<p>The Number of Contact per Residue (NCPR) for each domain of NpAS and DgAS.</p

Estimated folding free energies (kcal·mol⁻¹) for NpAS and DgAS.

a<p>H-bond energy for the backbone-backbone type.</p>b<p>H-bond energy for the sidechain-backbone and sidechain-backbone type.</p>c<p>Electrostatics energy contributions of the charged pairs.</p>d<p>Energy contribution by conformational entropy at room temperature.</p>e<p>Helix dipole (mainly) and others.</p

Virtual ala-scan for the seven additional glycine residues of DgAS.

<p>Virtual ala-scan for the seven additional glycine residues of DgAS.</p

Comparison between the structures of NpAS and DgAS.

a<p>The distance and the angle cutoffs used for the calculation of H-bonds are 3.0 Å and 150°, respectively. The first number of the content in the bracket is the count of backbone-backbone H-bonds, the second one gives the count for the sidechain-sidechain/backbone H-bonds.</p>b<p>The distance cutoff used for the calculation of salt bridges is 3.5 Å.</p

The structure based sequence alignment for NpAS and DgAS.

<p>The conserved residues are highlighted by red background.</p

The distribution of proline residues among each domain.

<p>The distribution of proline residues among each domain.</p