Construction and identification of the expression vector
According to the protein sequence of DIA (GenBank: CP002293.1) in NCBI, the gene sequence was codon optimized for the preference of E. coli. The optimized gene was synthesized, and cloned into the pUC57 vector. The rDIA fragment was double-digested using NdeI/XhoI and sub-cloned into the pET28a expression vector. Schematic of the construction of recombinant expression plasmid pET28a-rDIA was shown in Fig. S2A. The recombinant expression vector was identified by PCR using the primer pairs of T7-F (5’-TAATACGACTCACTATAGGG-3’) and T7-R (5’-GCTAGTTATTGCTCAGCGG-3’). As shown in Fig. S2B, the target fragment of rDIA was shown to be approximately 1000 bp, which is almost identical to the theoretical value. We also identified the pET28a-rDIA plasmid by gene sequencing from GENEWIZ Inc. (Suzhou, China).
Expression and purification of the rDIA
The flow chart of expression and purification process of rDIA was shown in Fig. 1A. As shown in Fig. 1B, the target protein band was found in both bacterial lysate of BL21-rDIA and supernatant in TB culture, which indicated that the rDIA was successfully soluble expressed. Figure 1B also indicated the molecular mass of recombinant rDIA consistent with deduced molecular mass of 29 kDa. Large scale protein expression was performed under the auto-induction fermentation medium. After purified by using HisTrap FF column, the protein samples were identified using SDS-PAGE (Fig. 1C). The rDIA was over 95% pure and with a yield of 110 mg/L culture. The molecule weight of rDIA was similar to the thermophilic alcohol dehydrogenase (htADH) (Liang et al. 2004), an intracellular diaphorase, which was isolated from a strain of Bacillus stearothemophilus with the molecular weight of 30 kDa.
Enzymatic characterization of rDIA
As the temperature have great influence on the enzymatic reaction, the optimum temperature of purified rDIA was determined by measuring its activity at a series of temperatures of 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 75, and 80°C at pH 6.5. As shown in the Fig. 2A, rDIA displayed maximum activity when the temperature at 55°C, and maintained good catalytic activity at broad temperature range from 30°C to 80°C. Nearly 60% of the maximum activity was remained when the temperature increased to 80°C. We also measured the thermostability of rDIA by heating the enzyme at different temperatures for 40 min before the measurement. The rDIA showed good activity at temperatures of 30–60°C (Fig. 2B), and nearly 85% enzymatic activity was still remained after treated at 60°C. These results indicated that the optimum temperature of this rDIA was 55°C and possessed outstanding thermostability from 30–60°C. rDIA showed more thermal stability than DLD, which has been heterologous expressed and purified from E. coli with a sharp decrease in enzyme activity above 30°C and completely inactivated at 70°C (Kianmehr et al. 2017).
The effects of pH on the rDIA activity were evaluated after exposure to pH ranged from 5.0 to 9.5 at 37°C, and the results were shown in Fig. 2C. The enzyme activity in all pH conditions were all above 40%, and the optimum pH was 6.5. Besides, the stability of this enzyme at different pH was probed by treating the rDIA with different pH buffer ranging from 3 to 12 at 4°C for 6 h before the enzymatic measurement (Fig. 2D). The rDIA showed excellent pH stability with a broad range from 5.0 and 9.0, which remain over 85% activity. Even at pH 10.0, the enzyme maintained approximately 60% catalytic activity. These results proved that the optimum pH of rDIA was 6.5, and possessed good pH stability. In this study, the optimum pH and stability of the rDIA were similar to rBfmBC, encoded by Clostridium kluyveri rBfmBC gene (Chakraborty et al. 2008). And the optimum pH of the recombinant enzyme rBfmBC was found to be about 7.0, while it showed high activity only in a narrow pH range of 6.0 to 8.0 in diaphorase assays (Chakraborty et al. 2008).
We also probed the effect of different mental ions and chemical compounds on the enzymatic activity of rDIA. The rDIA (5 U/mL) was dissolved in 0.1 M potassium phosphate (pH 6.5) was incubated with each chemical at 37 ℃ for 30 min, and the catalysis activity were measured. As can be seen in Table 1, all of the regents showed negligible effect on the enzyme activity, which was similar to the results of other diaphorases (Kianmehr et al. 2017; Dietrichs et al. 1990), which indicated that the rDIA presented promising stable enzymatic property. Moreover, to calculate the Michaelis constant, the substrate NADH was diluted with 2 times gradient to determine the activity of diaphorase at different concentrations of NADH. The kinetic curve of rDIA was drawn based on GraphPad prism 6 software. The results calculated by software of Michaelis constant showed Km = 0.09 mM and Vmax = 81.40 µM/min (Fig. 2E).
Table 1
Effects of various chemicals on rDIA activity. The enzyme activity without any reagents was defined as 100%, and results represent means ± SD (n = 3).
Reagents | Concn. (mM) | Residual activity (%) |
None | — | 97.3 ± 3.5 |
Ca2+ | 2.0 | 96.8 ± 3.4 |
Zn2+ | 2.0 | 99.2 ± 2.3 |
Fe2+ | 2.0 | 102.4 ± 2.7 |
Mg2+ | 2.0 | 98.5 ± 3.9 |
NaN3 | 2.0 | 107 ± 1.8 |
PMSF | 2.0 | 97.8 ± 3.4 |
EDTA | 2.0 | 97.3 ± 3.5 |
Comparation of rDIA with commercial diaphorase
We compared the enzymatic property of the rDIA with commercial diaphorase by using the β-hydroxybutyric acid test kit (Abbexa, UK). The results showed that the enzymatic activity of rDIA was similar to the commercial diaphorase (Fig. 3A). Moreover, we evaluated the enzyme stability of rDIA and commercial diaphorase by storing at 4°C and 37°C for 14 days. As shown in Fig. 3B, the enzymatic activity of rDIA was similar to the commercial enzyme after stored at 4°C, while significantly higher than the other one at 37°C, which indicated the rDIA was more stable than commercial enzyme.
Structure changes and conformations transmission of rDIA at different pH were probed by MD simulations
As the pH was important in the application of diaphorase, we investigated the structure and conformation transitions of rDIA at different pH solutions by using homologous modeling and MD simulations. Firstly, the Cα-RMSD and atom-atom contacts of the whole protein were used to represent the enzyme activity at two pH values of 3.0 and 6.5. Currently, Bacillus smithii’s FMN-dependent NADH-azoreductase (PDB code: 6JXS, resolution: 1.95Å) has been crystallized, and has a sequence identity of 60.66% with rDIA (Fig. S3). The residue completeness and crystal structure resolution were also proved FMN-dependent NADH-azoreductase could be considered as the homology model of rDIA in the following MD simulations.
The Cα-RMSD and atom-atom contacts were displayed as a function of time in Fig. 4. As shown in Fig. 4A, the Cα -RMSD value at pH 3.0 was higher than pH 6.5 during the last 25 ns. And atom-atom contacts at pH 6.5 was generally larger than pH 3.0 during the whole 50 ns (Fig. 4B). Therefore, the structure of rDIA was more stable at pH 6.5 than pH 3.0 based on the results of the MD simulation.
We also analyzed the changes of RMSF, which represented the flexibilities of the individual residues in the protein (Sun et al. 2016), at pH 3.0 and 6.5. Time dependence of the RMSF values of Cα atoms of rDIA were calculated and displayed as shown in Fig. 4C. Two residue regions of 125–246 and 150–170 present higher RMSF values at pH 3.0 than 6.5, indicating the structure of these two regions were flexible and easily influenced by the pH changes. Of note, majority residues in 125–246 and 150–170 regions were negatively charged, we speculated that the negatively charged residues might contributed most in the rDIA structure changes at different pH.
The changes of secondary structures of rDIA at pH 3.0 and pH 6.5 was also investigated. The total contents of secondary structures of rDIA during the simulations at pH 3.0 and 6.5 were similar, but in the 141–161 domain, there were obvious differences in the secondary structure composition between these two pH (Table 2). Real time simulations of the secondary structure changes 50 ns at pH 3.0 and 6.5 were also monitored in Fig. 5. Of note, in the 141–161 domain that labeled by black wire frame, the structure of helix changed into turns with the simulation time under the condition of pH 3.0 (Fig. 5A), while there were not significant changes in the same fragment at pH 6.5 (Fig. 5B). It was indicated that the secondary structures of rDIA easily changed at pH 3.0. Representative snapshots of rDIA at pH 3.0 and pH 6.5 values (Fig SI 4) also proved that pH can affect the structure of rDIA.
Table 2
Average content of secondary structures of complete sequence and fragment 141–161 of rDIA at different pH values.
Secondary structures | Helix (%) | Coil (%) | Turn (%) | β-sheet (%) | 310 - helix (%) |
Total | pH 3.0 | 42.36 | 24.75 | 10.89 | 21.66 | 0.34 |
pH 6.5 | 42.93 | 23.83 | 11.45 | 21.24 | 0.55 |
141–161 domain | pH 3.0 | 1.76 | 41.70 | 18.91 | 33.10 | 4.52 |
pH 6.5 | 0 | 36.87 | 27.66 | 33 | 2.46 |
Mutation prediction of rDIA by MD simulations
The above results proved that pH can affect the structure of rDIA through different charged states of amino acids, especially of the 141–161 domain. Based on these results, we furtherly investigated the key residues on maintaining the enzymatic stability by using MD simulations, which was helpful for the direction evolution to improve the enzyme activity and stability. According to the homology modeling and MD simulations, the binding sites were formed from positions of 17–20, 52–60, 101–105, 146–150, 172, 186–187, which were related to enzyme catalysis pocket (Fig. 6). In order to improve the binding affinity, we did alanine-scanning mutagenesis for activity domain of rDIA and screened out the amino acid positions that had great energy changes, and then made site directed full mutation scanning of these amino acids. F105, M186 and W60 were found that there was a large energy change after alanine-scanning mutagenesis. Then site directed full mutation scanning was carried out for F105, M186 and W60, and the energy of F105W, F105R and M186R was negative (Fig. 6A), which indicated that the energy was reduced. Due to the reduction in energy, these mutants were able to improve the binding affinity of rDIA (Fig. 6B). We did mutation-scanning far away from the activity domain to screen for stability mutations under the optimal pH 6.5. The results of mutation-scanning showed that the energy change of A2Y, P35F, Q36D, N210L, F211Y was negative, especially A2Y and Q36D (Fig. 6C). Due to the reduction in energy, these mutants were able to improve the stability of rDIA (Fig. 6D). In summary, F105W, F105R and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.