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.