2.5.3 Interaction analysis
Interaction analysis of the conformations in the simulations of wild type and the mutant shows that when the alanine at position 355 was mutated to asparagine (Fig.3A and 3B), the amino acid side chain of asparagine was extended. Due to the effect of steric hindrance, Ser356 was pushed forward. Moreover, water bridge was formed between substrate p NPR and Ser356, which strengthens the hydrogen bond interaction between the enzyme and the substrate, thereby increasing the activity of the enzyme on the substrate. Thus, these results were in conformity with the results of the docking MD simulation studies of previous reports, in which the formation of hydrogen bonds increases the activity of the enzyme on the substrate.46, 52 When the serine at position 356 was mutated to tyrosine (Fig.3C and 3D), a π-π stacking was formed between the phenyl of tyrosine and the nitrophenyl ring of the substratep NPR, which strengthens the interaction force between the enzyme and the substrate, and bring out improved enzyme activity. The correlation between the pi-pi stacking and the strength of the substrate-enzyme complexes is also seen in a coplanar mu-S bridged 1,10-phenanthrolinepalladium(II) dinuclear complex. 53
As for mutant D525N, when aspartic acid at position 525 was mutated to asparagine, as shown in Figure 3E and 3F, i.e. the acidic negatively charged amino acid was mutated to a polar uncharged amino acid, the negative charge in the vicinity of Asn525 suddenly changed to positive charge. The strong change of local environmental charge led to a large change in the enzymatic properties, and could justify the increase of both enzyme activity and thermostability, as results from Naito et al.54 that the change in the charge of the catalytic domain led to the increase of the activity and thermostability of the enzyme.
Thermostable enzymes with favorable catalytic efficiency are very limited in nature and hard to obtain. Therefore, how to effectively improve the thermal stability and catalytic efficiency of enzymes has always been a hot issue in research. In most literature reports,28-31, 55 it is shown that the increased flexibility of the enzyme structure can improve the catalytic activity of the enzyme, and the increased rigidity of the enzyme structure can improve the thermostability of the enzyme. Based on the similar designing ideas, Sniha et al28, Humer et al25 and Ashraf et al26 have demonstrate that it is impossible to use the corresponding rational or semi-rational design to achieve the goal of enzyme engineering, that is, to improve the catalytic activity and thermostability of the enzyme at the same time. Here, instead of adjusting the global rigidity, we selected mutation sites in the catalytic domain near the substrate binding, modifying only the local rigidity without changing the overall structure of the enzyme, and used a double screening strategy, successfully obtaining mutants with improved catalytic activity as well as thermostability. By using the semi-rational design procedure, the mutation at Asp525 , which is located within the range of 5 Å of the catalytic domain bound to the substrate (Fig. 1A and 1B), were shown effective in simultaneous improvement of the catalysis activity and thermal stability. Th results indicate the new semi-rational strategy is very effective to improves both enzyme activity and thermostability. In addition, our study suggests that the 5 Å range around the substrate binding site is the effective candidate to conduct protein engineering mutation for improve enzyme activity and thermal stability.