| Aspartate Kinase(AK)is the first key speed-limiting allosteric enzyme of aspartate(L-Asp)in the synthesis of aspartate family amino acids.However,its catalytic activity is affected by the synergistic feedback inhibition of the terminal products lysine(Lys)and threonine(Thr),the aspartic acid biosynthetic metabolites cannot accumulate excessively.Therefore,weakening or removing the feedback inhibition in the biosynthesis process will improve AK enzyme activity and essentially increase the production of aspartate amino acids.In this study,the Corynebacterium pekinense aspartate kinase(Cp AK)is a monomer and selected for the study.The key residue positions around the Thr ligand were site-directed for saturation mutations.Then the mutant strains are screened by high-throughput screening technology,and the enzyme reaction kinetics as well as enzymatic properties of wild-type(WT)AK and mutant AK are studied respectively.Quantum chemical calculations were used to explore the detailed catalytic reaction mechanism of Cp AK.The specific mechanism of AK catalytic reaction would give a deeper understanding of the product feedback inhibition mechanism of AK,and it provides guidance for the design of new and efficient enzymes.The main findings are as follows:1.Determination of mutation sites.Through homologous sequence alignment and analysis of monomeric Cp AK,it is found that Asp292,Gly295,Glu296,Ala297,and Gln316 around the Thr inhibitor are the key residue positions and highly conserved.Site-directed saturation mutations were carried out to weaken or remove the feedback inhibition by the metabolic end products,increasing AK enzyme activity,and improve enzymatic properties.2.Construction of mutants.Site-directed saturation mutations were performed on Gly295 and Glu296 sites,and 4 single mutants with increased enzyme activity were obtained through digestion,transformation,and high-throughput screening: G295 A,E296H,G295 L and E296 Q.And on the basis of single mutants,double mutants G295A/E296Q(abbreviated as AQ)and G295L/E296Q(abbreviated as LQ)were constructed.On the basis of the quadruple mutants T379N/A380C/G171I/Y198N(NCIN)in the previous laboratory,the quintuple mutants T379N/A380C/ G171I/Y198N/D292Y(NCINY),T379N/A380C/G171I/Y198N/ G295L(NCINL),T379N/A380C/G171I/Y198N/E296Q(NCINQ),T379N/A380C/G171I/Y198N/A297K(NCINK),and T379N/A380C/G171I/Y198N/Q316P(NCINP)were constructed with combining single mutants D292 Y,G295L,E296 Q,A297K,and Q316 P.After induction and expression of each mutant strain,separation and purification,the crude enzyme solution and purified solution were verified by SDS-PAGE and Western blot,and it was found that all 11 mutants were successfully expressed in E.coli.3.Enzymatic reaction kinetic analysis.From the results of enzyme kinetics study of WT AK and mutant AK,it can be seen that the enzyme activities of single mutants G295 A,E296H,G295 L and E296 Q are 3.76,14.04,8.46,and 16.80 times higher than that of WT AK,respectively,and the Km value is reduced,indicating that after the mutation,the affinity of the enzyme and the substrate increased.The value of n decreased,indicating that the positive synergy decreased after the mutation.The enzyme activities of double mutants AQ and LQ were increased by 19.02 times and 23.20 times,respectively,the Km of AQ increased,and the value of n decreased,while the Km and n values of LQ both decreased.The enzyme activities of quintuple mutants NCINY,NCINL,NCINK,NCINP and NCINQ were 85.09,87.20,97.67,95.37,and 99.43 times higher than the wild type,and the Km value and n value were reduced.4.Characterization of enzyme properties.The optimal temperature of the single mutant strains G295 A and E296 Q were 26℃ and 30℃,respectively,and the optimal temperature of G295 L and E296 H was the same as that of the wild type,which was 25℃.The optimum temperature of the double mutants AQ and LQ are 26℃ and 30℃,respectively.The optimum temperature of the mutants has been increased,and the heat resistance has been significantly improved.The optimal p H of the single mutant strains G295 A,G295L,E296 H,E296Q are 7.5,8.0,8.5,8.5,and the half-lives are 4.3 h,6.0 h,4.7 h,7.1 h,respectively.The optimum p H values of the double mutants AQ and LQ are 7.5 and 8,and the half-lives are 4.2 h and 5.4 h,respectively.After the mutation,the optimal p H is lower than that of WT AK(8.0),indicating that the acid resistance is improved,and the half-life is greater than that of WT AK(4.7 h),indicating that the stability is enhanced.After mutation,the inhibitory effects of different concentrations of inhibitors on single mutants and double mutants are weakened,and even under the combination of different inhibitors,there is a significant activation effect.The five mutants NCINY,NCINL,NCINK,NCINP and NCINQ have optimum temperatures of 25℃,28℃,35℃,35℃,28℃,and optimum p H values of 8.5,8.5,7.5,8.5 and 8.0,and their half-lives is 6.7 h,5.2 h,6.1 h,5.2 h and 6.3 h,respectively.Inhibitors of different concentrations have varying degrees of activating effects on the five mutants,basically releasing Lys inhibition,partially releasing Thr inhibition and synergistic feedback inhibition.5.Investigation of the Catalytic mechanism research.We used the DFT/B3 LYP method in the Gauss16 program DFT/B3 LYP method to study the catalytic reaction mechanism of Cp AK in detail.The study found that as the reaction progresses,the P-O bond formed by P atom on the γphosphate group of ATP and O3 in the β phosphate group is broken.The γ phosphate is broken.The group vibrates back and forth between the branched O atom of Asp and the O3 atom of ADP to form a transition state structure;after that,the γ phosphate group further approaches the substrate Asp and forms a bond with its branched O atom to form the final transfer product.The reaction of AK catalyzing the transfer of the γ phosphate group of ATP to the branched carboxyl group of the substrate Asp is an endothermic process.In this process,the amino acid residues of Cp AK have the functions of balancing the charge,stabilizing the structure,and reducing the reaction energy. |
