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.