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.