3.4. Energy and interaction analysis of designed enzymes
Molecular dynamics simulations (MDS) of designed enzyme were carried out. Energy decomposition analysis of mutation was studied by data obtained from MDS. Co-evolved residues pairs were mutated in order to form new and strong salt bridges. Electrostatic interaction energy and total interaction energy was calculated for further selection of mutated pairs (Table 4), which indicated that electrostatic interaction energy contribute to the most part of interaction energy. Buried interactions network can enhance interfacial interactions of subunits and enzyme stability [27].
The benefits of considering dynamic conformation change for enzyme design can also enable engineering enzymes from the prospect of both structure and function[28]. The structure of the enzyme undergoes dynamic conformational adjustments with increasing temperature, resulting in gradual loosening of the unstable secondary structure and consequently disfolding. The introduction of interfacial hydrogen bonds contributes to the thermostability of the enzyme[29]. Energy provided by each hydrogen bonds is about 0.6 kcal/mol [30]. MDS also indicated that the average number of hydrogen bonds of wild-type DAwild is 108. Therefore, there are 8 extra hydrogen bonds of DA11 than DAwild, which provided extra energy barrier for unfolding (Table 5).
The average number of salt bridges of DAADHs were calculated by MDS result with 0.1 M NaCl. Salt bridges networks play an important role in maintain the rigidity of the three-dimensional structure of enzyme. Formation of more salt bridges inside the protein molecular is beneficial to maintain the structural stability[31]. The average number of salt bridges of DA06 and DA11 are 1.4 and 1.2-fold of that of DAwild, respectively. This result indicated that increased salt bridges and hydrogen bonds enhanced structural rigidity and improved thermostability.
Therefore, DA06 and DA11 were selected for further experiment study. The formation of a large number of non-covalent interactions within the modified enzyme is beneficial to the thermostability, which has been strengthened by multipoint mutations.