Study On Spatial Modification Of Catalytic Active Sites And Allosteric Regulation Mechanism Of Aspartate Kinase From Corynebacterium Pekinense

Aspartate kinase(AK)is the first key rate-limiting enzyme of the biosynthesis of aspartic acid family amino acids in organisms,and simultaneously inhibited by the synergistic feedback of downstream metabolites threonine(Thr)and lysine(Lys).The downstream carbon flow competition increases and the supply of methionine(Met)synthesis decreases,which limits the accumulation of terminal product Met.In this study,through the structural analysis of aspartate kinase from Corynebacterium pekinense(Cp AK),the key residual sites around the catalytic centers ATP and Asp were selected for site-specific saturation mutagenesis,and the mutants with significantly increased enzyme activity were obtained through high-throughput screening.The enzymatic kinetics and characterization of enzyme properties of Wild-Type(WT AK)and mutant AK were studied,combined with molecular dynamics simulations to further explore its allosteric regulation mechanism.The results are as follows:1.Selection of the mutation site.Through the structural model and homologous sequence alignment analysis of Cp AK,the key residual sites Gly27,Gly28,Ser29,Ser227 and Ile229 around the catalytic active center ATP were selected to superimpose saturated mutations based on the triple mutant strains T379N/A380C/G171I(NCI)obtained in the previous laboratory,and quadruple mutant was constructed.Continue to select Glu92,Ser172,Asp173,Asp193 sites,and superimpose mutations on the basis of quadruple mutant with excellent traits to obtain quintuple mutant,with significantly improved enzyme activity.2.Construction of mutant.On the basis of triple mutant NCI obtained in the early stage of the laboratory,through designing mutant primers,PCR amplification and host transformation and other steps,three quadruple mutants with increased enzyme activity were obtained by high throughput screening technique,which are T379N/A380C/G171I/S29 R,T379N/A380C/G171I/S227 N and T379N/A380C/G171I/S227D(referred to as NCIR,NCIN and NCID).On the basis of the mutant NCID with significantly increased enzyme activity,combined with the current laboratory research results,quintuple mutant strains T379N/A380C/G171I/S227D/E92 V,T379N/A380C/G171I/S227D/S172 P,T379N/A380C/G171I/S227D/D173 G,T379N/A380C/G171I/S227D/D173 R,T379N/A380C/G171I/S227D/D193T(referred to as NCIDV,NCIDP,NCIDG,NCIDR,NCIDT))were constructed.SDS-PAGE and Western Blot methods verified that all the mutants were successfully heterologous expressed in E.coli.3.Study on enzymatic kinetics.The enzymatic kinetics of wild type and mutant was studied.The results showed that the maximum catalytic rate Vmax of each mutant was significantly higher than that of wild type AK.Among them,four mutant strains NCIR,NCIN,NCID were 71.99,76.88 and 80.41 times higher,and that of quintuple mutants NCIDV,NCIDP,NCIDG,NCIDR and NCIDT increased by 82.20,83.71,86.33,90.76 and 82.43 times,respectively.At the same time,the Km value was lower than that of the wild type AK,indicating that the affinity of the substrate was increased,which was beneficial to the binding with the substrate.4.Characterization of enzymatic properties.The optimum temperatures of the quadruple mutant NCIR,NCIN and NCID AK were 28 ?,30 ? and 30 ?,and the optimum temperatures of the quintuple mutants NCIDV,NCIDP,NCIDG,NCIDR and NCIDT AK were28 ?,26 ?,30 ?,28 ? and 26 ?,respectively,which were significantly higher than those of the WT AK(the optimum temperature was 25 ?).The results indicated that the heat tolerance was improved.The optimum p H values of the quadruple mutant NCIR,NCIN and NCID AK were 9.0,8.5,8.5,respectively,and the optimum p H of NCID AK was 0.5 lower than that of the NCI AK(the optimum p H was 9.0),and the acid tolerance was enhanced.The optimum p H of the quintuple mutants NCIDV,NCIDP,NCIDG,NCIDR and NCIDT AK were8.5,8.5,8.0,7.5 and 9.0,respectively,while the optimum p H of NCIDR was 0.5 lower than that of the WT AK(the optimum p H was 8.0).The acid resistance is obviously enhanced,which is beneficial to the production and fermentation of engineering bacteria.The half-life of WT AK was 4.9 h,the half-life of NCI AK was 3.1 h,the half-life of quadruple mutant is 3.0 h,3.6 h,3.9h,and the half-life of quintuple mutant is 3.6 h,4.1 h,4.3 h,4.6 h,3.1 h,respectively.Compared with WT AK,the half-life of each mutant is shorter,while the half-lives of quadruple mutant NCIN,NCID and quintuple mutants NCIDV,NCIDP,NCIDG and NCIDR were longer than that of triple mutants.In the presence of different concentrations and different combinations of inhibitors,the enzyme activity of each mutant was increased,the inhibition was weakened,and even activated,and showed obvious concentration dose dependence.Under the condition of different combinations of 10 mmol/L Thr+Met,Lys+Met and Thr+Lys+Met,each mutant showed obvious activation effect,especially under the condition of 10 mmol/L Thr+Lys+Met.The relative enzyme activities of the quadruple mutants were 130.46%,135.12% and 143.35%,respectively,while those of the quintuple mutants were 139.74%,156.22%,150.81%,156.48%and 144.22%,respectively.5.Allosteric regulation mechanism.The molecular dynamics simulation results showed that in the absence of inhibitors,the distance between the P atom of the ? phosphate group of ATP and the carboxyl O atom of the substrate Asp in the WT system was 2.76 (?),and the catalytic reaction can take place.ATP and the substrate Asp binding domain formed hydrogen bonds with Ser29,Asp193,Arg203,Tyr198 and other residues of AK,which played a role in stabilizing the catalytic substrate,and it is beneficial to the occurrence of the reaction.when the inhibitor Lys and Thr were combined,the conformation changeed and the effective residual domain moved relatively,the distance of P-O increased,and the hydrogen bonding force was destroyed,which weakened the binding force between AK and ATP and substrate Asp,destroy the stability of the system,and seriously reduced the catalytic efficiency of AK.After mutation,the side chain of the mutation residue extended,which lead to the distance between ATP shortened.The distance between the P atom of the ? phosphate group of ATP and the carboxyl O atom of the substrate Asp was reduced to 2.70 (?).At the same time,the hydrogen bond was formed between the mutant residue and ATP,and the electrostatic interaction increased to-292.26 kcal/mol.the binding of ATP to AK was stable,which was beneficial to catalysis.Therefore,mutagenesis of AK can effectively weaken the allosteric regulation of metabolites.