3.5 Structure analysis of mutant enzymes for improving
thermostability
To analyze the conformational change of the mutations caused by each
substituted residue, three-dimensional structure of WT and mutants were
homology modeled with Swiss-Model protein automated modeling
program.
The tight packing of protein interiors plays a vital role in protein
stability for the burial of both polar and nonpolar groups, and one
-CH2- group is buried on folding contributes 1.1 ± 0.5
kcal/mol of energy to protein stability.[34] As
shown in Figure 4A, a single -CH2- group was added to
side chain after mutating the amino acid Val to Leu at position 280.
This seems to reveal that the introduction of alanine’s bulky non-polar
side chain may be responsible for improving the stability. In addition,
inspection of the structure model of the V280L showed that L280 were
located in the ɑ-helix (Figure 4B), and V280L substitution also
generated two Vander Waals forces (VDW) bonds with Q329 and T330 of the
other ɑ-helix. Therefore, the two newly introduced VDW may also
stabilize the local stability, thereby facilitate the geometry more
stable. The same strategy was also applied to improve the
thermostability of Bacillus thermoleovorans pullulanase
successfully.[20] More interestingly, we were
surprised to find that all the amino acids at 280 sites from other
sources were L except for V from the Pantoea dispersa UQ68J
through multiple sequence alignment Figure 4C. Therefore, the
improvement of thermostability of V280L mutant may also be related to
the evolutionary conservatism of the enzyme.[35]
Previous studies have pointed out that molecular interactions, including
hydrogen bonds, disulfide bonds, VDW, aromatic–aromatic interaction,
and hydrophobic interaction are the major structural factors that take
effect on the protein thermostability. In all of these factors, the
contribution of hydrophobic interaction to protein stability accounts
for about 60%.[36] As shown in Figure 4E, a new
hydrophobic network formed for substituted residue from hydrophilic S
(Figure 4D) to strong hydrophobic F at the site 499, which contains four
residues (P24, W339, P495, and L519) within 5 angstroms. Therefore, the
residue 499 greatly changed the hydrophobic stacking around the
mutation, thereby enhancing the hydrophobic interaction effect. In
addition, a cation–π interaction between F499 and W339 was found after
mutation, which may further improve the thermostability of mutant S499F.
In summary, the improved thermostability of mutant S499F may be the
result of the hydrophobic interactions and cation–π interaction.
Moreover, there were no new molecular interactions being introduced into
the double mutant V280L/S499F (Figure 4F), maybe the synergistic effect
of these two single point mutations further promoted the improvement of
stability of the double mutant.