2.3 Secondary structural characteristics of mutants with better catalysis efficiency and thermostability
In order to further explore the reasons why mutants A355N, S356Y and D525N with improved enzyme activity have two different characterizations for thermostability, far-UV CD was used to investigate the effect of site-directed mutations on protein structure. The result showed that the CD spectra of both WT and mutants were nearly 100% superimposed (Table S2). As showed in Fig. S3, both r-Rha1 and three mutants presented a negative peak at 205-245 nm a typical feature for α-helices, where the strong positive peak at 185-195 nm represented a typical signature of β-sheets. The secondary structure of r-Rha1 and the mutants A355N, S356Y and D525N were calculated based on the CD spectra. Slight decreases of α-helix and β-turn were observed in the three mutants, whereas β-sheet and random were increased. Specifically, the content of α-helix of WT, A355N, S356Y and D525N was 27.5%, 25.3%, 25.3% and 25.7% while the content of β-sheet was 26.6%, 27.6%, 29.7% and 28.6%, respectively. Meanwhile, the β-turn component decreased by 0.7%, 0.4% and 0.6% in mutants A355N, S356Y and D525N, respectively. In Fig. S4, theTm value of the mutant A355N was almost unchanged compared to the wild type: Tm of S356Y was 0.73 times that of wild type while D525N was 1.1 times. This is consistent with what Mohd et al48 have showed, i.e. the substitutions of one or a few amino acids, which lead to the improvements in thermostability and optimum temperature of the mutants did not cause a substantial change in the secondary structures of the enzymes. This means that the change of a single amino acid does not cause major changes in the secondary structure, and the difference of thermostability among these three mutants could not be attributed to the change of the secondary structure.