Insight into the structure, dynamics and the unfolding property of amylosucrases: implications of rational engineering on thermostability.
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 Deinococcus geothermalis amylosucrase (DgAS) is more stable than Neisseria polysaccharea 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.
Beijing Institute of Biotechnology, Beijing, China.
Ming
M
Liu
Shuang
S
Wang
Tingguang
T
Sun
Jiguo
J
Su
Yuanxing
Y
Zhang
Junjie
J
Yue
Zhiwei
Z
Sun
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D017124:Conserved Sequence; D034301:Deinococcus / Q000201:enzymology*; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry*; D006860:Hydrogen Bonding; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009343:Neisseria / Q000201:enzymology*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D013816:Thermodynamics
D005964:Glucosyltransferases; C019700:amylosucrase