ATP Binding Site Spatial Modification And Characterization Of Enzymatic Properties Of Aspartate Kinase From Corynebacterium Pekinense

Aspartate Kinase(AK)is the first key allosteric enzyme to catalyze the synthesis of amino acids in the aspartate family.It is inhibited by the synergistic feedback of the metabolites Lys and Thr.Resulting in difficulties of accumulating a large number of products in the downstream of this synthetic pathway.This study aims to increase AK enzyme activity and release feedback inhibition by site-directed mutation.After obtaining the monomer aspartate kinase from Corynebacterium pekinense and analyzing the structure,the key residue sites around ATP were selected for site-directed mutation.Mutations with increased enzyme activity were obtained through high-throughput screening.Enzymatic kinetic analysis and characterization of wild-type and mutants AK were studied.The results were as follows:1.Mutation sites screening.Tyr198,Asp201,Val276,and Thr278 were selected as mutation sites through homologous sequence comparison and Cp AK model analysis.2.Mutants construction.Seven single mutants were obtained through site-directed mutations on selected mutation sites and high-throughput screening,which were Y198 N,Y198D,D201 M,D201R,V276 L,V276A,T278 K.Based on the single mutants and three mutants obtained in the laboratory,double mutants Y198N/D201 M,Y198N/D201R(referred to as MN and RN)and four mutants T379N/A380C/G171I/Y198 N,T379N/A380C/G171I/D201M(referred to as NCIN,NCIM)were constructed.SDS-PAGE and Western blot confirmed that all mutants were successfully expressed in E.coli.3.Enzymatic kinetic analysis.Enzyme activity were increased of seven single mutants,compared to wild-type AK increased 15.25,11.11,13.55,12.81,6.46,3.29,7.06 times respectively.Only the Km value of V276 A increased,the Km value of other mutants decreased,and the affinity with the substrate was enhanced.The enzyme activities of double mutants MN and RN were increased by 18.26 and 16.08 times compared with wild type,and the Km value of RN was increased.The enzyme activities of the four mutants NCIN and NCIM increased by 75.64 and 68.50 times,respectively,the Km values decreased,and the affinity with the substrate was enhanced,the n values of all the mutants decreased,and the positive synergy were reduced.It indicates that the affinity of the enzyme with the inhibitor decreases after binding to the substrate.4.Analysis of enzymatic properties.The optimum temperature of wild-type AK was 25°C,the optimum p H was 8,and the half-life was 4.6 h.The optimum temperature of single mutants Y198 N,Y198D,D201 M,D201R,V276 L,V276A,and T278 K were 26°C,28°C,25°C,26°C,26°C,25°C,and 28°C,respectively.And the optimum temperature of Y198 D and T278 K were 3°C higher than that of wild type.The optimum p H of T278 K was 7.5,which was beneficial to fermentation production of strains.The optimum p H of Y198 D was 8.5,and the optimum p H of the other mutants was 8,which was the same as the wild type.The half-lives were 4.6 h,3.8 h,4.1 h,3.2 h,4.3 h,5 h,5.5 h.The half-lives of V276 L and T278 K were significantly longer than those of wild type.The optimum temperature of double mutants MN,RN and four mutants NCIN and NCIM were 28°C,26°C,25°C,26°C and high temperature resistance of MN was enhanced.The optimum p H of MN and NCIM was 7.5,and the tolerance p H range of the enzyme was widened.The half-lives were 5.3 h,5.9 h,3.9 h,and 4.2 h,respectively,and the half-lives of MN and RN were extended by 0.7 and 1.3 h.With the exception of V276 A,the enzyme activity of all mutants was increased to varying degrees in the presence of inhibitors at different concentrations,the inhibitory effects were weakened,and even activation was shown in the presence of inhibitors at different concentrations.In particular,four mutants in the presence of different concentrations of Lys,the inhibitory effect was completely released.Under 10 mmol/L Lys+Thr+Met conditions,the four mutants NCIN enzyme activity reach 140.62 mmol/L,and the NCIM enzyme activity can reach 142.20 mmol/L.5.Analysis of protein spatial structure.The number of hydrogen bonds increased between 198 site and ATP after 198 site mutation developed and generated Asn198 and Asp198.The force between the 201 site and ATP was increased after 201 site mutation developed and generated Met201 and Arg201,while the distance between the 201 site and ATP was shortened.The electrostatic potential of the protein increased after 276 site mutation developed and generated Ala276 and Leu276.The side chain of 278 site increased after 278 site mutation developed and generated Lys278,and the increased interaction with the surrounding residues Leu412 and Ala416 stabilized the ATP binding,which facilitated the catalytic reaction and increased the enzyme activity.