The enzymatic activity was enhanced by Ni2+ and ethanol. lysine-binding site in the regulatory region of CgAK performs a vital function in AK allosteric inhibition [16,17]. Open in a separate window Physique 1 Multiple sequence alignment of aspartate kinase (AK) with other members. CpAK from [18]. Open in a separate window Physique 5 Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the recombinant AK and its mutants. (a) Native PAGE of the recombinant AK and the mutants. M: molecular weight marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; and (b) SDS-PAGE of the recombinant AK and the mutants. M: high-molecular weight protein marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; lane 5: supernatant of induced sample; and lane 6: Western blot of the purified AK. 2.4. Kinetic Assay of the Wild Type (WT) and AK Mutants As shown in Table 1, kinetic parameters, namely, was obtained from Novagen (Madison, WI, Cgp 52432 USA). The recombinant plasmid pET-28a-AK was provided by our laboratory. 3.2. Construction of Mutant Strains The genomic DNA of was isolated with a genomic DNA extraction kit. The aspartokinase gene was then amplified by PCR, ligated to plasmid PMD 18-T, and then transformed to DH5. The plasmids were extracted and sequenced. After digestion with the restriction enzymes, namely, BamHI and (PDB ID 3aaw sequence identity, 99%) was used as the template protein. The BLAST was used for searching, and Swiss Model was used to build the 3D structure Cgp 52432 [31,32,33]. The distance between the residue of 169 and E92 was calculated with the program PyMOL (http://pymol.sourceforge.net/) for further structural analysis of WT and mutant proteins. 3.8. Molecular Docking The substrate and ATP were docked to the homology modeled AK [10] by using the Lamarckian Genetic Algorithm provided by AutoDock 4.2 software [28,34]. A cubic box was built around the protein with 36 ? 36 ? 36 ? points. 3.9. Molecular Dynamics (MD) Simulation and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) Calculations Eleven 10 ns structures of the complex were used as starting points for calculations of binding free energy. All simulations were performed using the Amber 11 package for 10 ns, with the amber 99 sb as the field-force parameter [25]. Binding free energies were calculated using the MM-PBSA method [35]. In addition, the two substrates used in the present study are highly comparable. According to previous studies [36,37], the entropy differences should be minimal such that the correlation between the experimental value and the calculated binding free energy may not be substantially improved. Therefore, the solute entropy term was neglected in the present study. For each MD-simulated complex, we calculated the is a member of the AK superfamily. Experimental data showed that this optimum temperature and pH of AK were 26 C and pH 7, respectively. The half-life was 4.5 h under the optimum conditions, and ethanol and Ni2+ strongly increased the enzymatic activity of CpAK. The steady-state kinetics study confirmed that AK is an allosteric enzyme, and enzymatic activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. The results of molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) showed that this residue Arg169 participated in substrate binding, catalytic domain name, and inhibitor binding. These findings can be used to develop new enzymes and provide a basis for amino acid production. Acknowledgments Funding for this work was provided by the national 863 plan project (No. 2013AA102206), the authors also would like to thank Jilin Provincial Science & Technology Department for supporting key project (No. 20130101139JC) and key project (No. 20150519012JH). Supplementary Materials Click here for additional data file.(835K, pdf) Supplementary materials can be found at http://www.mdpi.com/1422-0067/16/12/26098/s1. Author Contributions Weihong Min conceived and designed the experiments. Huiying Li performed the Cgp 52432 experiments. Huiying Li and Chunlei Liu analyzed the data. Weihong Min, Hongmei Li, and Jingsheng Liu provided reagents/materials/analysis tools. Weihong Min and Huiying Li wrote the paper. Conflicts of Interest The authors declare no conflict of interest..The enzymatic activity was enhanced by ethanol and Ni2+. lysine-binding site in the regulatory region of CgAK performs a vital function in AK allosteric inhibition [16,17]. Open in a separate window Physique 1 Multiple sequence alignment of aspartate kinase (AK) with other members. CpAK from [18]. Open in a separate window Physique 5 Native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the recombinant AK and its mutants. (a) Native PAGE of the recombinant AK and the mutants. M: molecular weight marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; and (b) SDS-PAGE of the recombinant AK and the mutants. M: high-molecular weight protein marker; lane 1: purified recombinant R169Y; lane 2: purified recombinant R169P; lane 3: purified recombinant R169D; lane 4: purified recombinant R169H; lane 5: supernatant of induced sample; and lane 6: Western blot of the purified AK. 2.4. Kinetic Assay of the Wild Type (WT) and AK Mutants As shown in Table 1, kinetic parameters, namely, was obtained from Novagen (Madison, WI, USA). The recombinant plasmid pET-28a-AK was provided by our laboratory. 3.2. Construction of Mutant Strains The genomic DNA of was isolated with a genomic DNA extraction kit. The aspartokinase gene was then amplified by PCR, ligated to plasmid PMD 18-T, and then transformed to DH5. The plasmids were extracted and sequenced. After digestion with the restriction enzymes, namely, BamHI and (PDB ID 3aaw sequence identity, 99%) was used as the template protein. The BLAST was used for searching, and Swiss Model was used to build the 3D structure [31,32,33]. The distance between the residue of 169 and E92 was calculated with the program PyMOL (http://pymol.sourceforge.net/) for further structural analysis of WT and mutant proteins. 3.8. Molecular Docking The substrate and ATP were docked to the homology modeled AK [10] by using the Lamarckian Genetic Algorithm provided by AutoDock 4.2 software [28,34]. A cubic box was built around the protein with 36 ? 36 ? 36 ? points. 3.9. Molecular Dynamics (MD) Simulation and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) Calculations Eleven 10 ns structures of the complex were used as starting points for calculations of binding free energy. All simulations were performed using the Amber 11 package for 10 ns, with the amber 99 sb as the field-force parameter [25]. Binding free energies were calculated using the MM-PBSA method [35]. In addition, the two substrates used in the present study are highly comparable. According to previous studies [36,37], the entropy differences should be minimal such that the correlation between the experimental value and the calculated binding free energy may not be substantially improved. Therefore, the solute entropy term was neglected in the present study. For each MD-simulated complex, we calculated the is a member of the AK superfamily. Experimental DLEU1 data showed that the optimum temperature and pH of AK were 26 C and pH 7, respectively. The half-life was 4.5 h under the optimum conditions, and ethanol and Ni2+ strongly increased the enzymatic activity of CpAK. The steady-state kinetics study confirmed that AK is usually.