Modulating Stereoselectivity Of L-Threonine Aldolase And Its Catalytic Mechanism Studies

Threonine aldolases（TAs）can catalyze aldol condensation reaction and its reverse reaction to synthesize b-hydroxy-a-amino acids with glycine as donor and aliphatic or aromatic aldehyde as acceptor.b-hydroxy-a-amino acids are important precursors of many agrochemicals and pharmaceuticals,playing an important role in the fields of medicine and fine chemicals.TAs can catalyze a one-step reaction to synthesize b-hydroxy-a-amino acids under mild reaction conditions,while its diastereoselectivity at C_bis very low,that is the“C_b”problem that has been receiving great attention.This problem seriously hinders the application of TAs in industrial production.In this study,the protein engineering of L-TA from Pseudomonas putida was performed based on amino acid sequence and structure information.The enzymatic activities of L-TA cleavage natural substrates L-threonine and L-allo-threonine have been improved by saturation mutation of key amino acid sites.At the same time,a high-throughput screening method of DNPH colorimetry based on its aldol condensation reaction was established,and the catalytic efficiency of it synthesize 4-methylsulfonylphenylserine was improved by combining saturation mutation and iterative combination mutation.In addition,the catalytic efficiency of it synthesize 4-methylsulfonylphenylserine was further improved by protein engineering through computer-aided design combined with triple code saturation mutagenesis strategies.The main results are listed as follows.（1）Mining,expression,purification and enzyme characterization of L-TAs.Based on the structural and functional domains of L-TAs,the genes encoding L-TAs in Pseudomonas putida KT2440,Pseudomonas protegens CHA0 and Pseudomonas putida PSALD were fished and the transformed into Escherichia coli BL21（DE3）for heterologous expression.The results of enzyme activity assay showed that the enzyme activities of KT2440,CHA0 and PSALD cleavage L-threonine were 5577,5407 and8465 U·mg^-1 respectively,and cleavage L-allo-threonine were 14311,8918 and 16814U·mg^-1 respectively.Both KT2440,CHA0 and PSALD reached the highest enzyme activity at p H 8.0 and were more stable in the range of p H 7.0-9.0.The optimum temperatures of KT2440,CHA0 and PSALD were 50,55 and 55°C respectively.In addition,both KT2440 and CHA0 have better tolerance to organic solvents in TBME,while PSALD has poorer tolerance to organic solvents.Mg²⁺and Ca²⁺can significantly promote their enzyme activities.（2）Studies on the matching of KT2440 traditional enzyme activity assay and product synthesis efficiency.Through molecular docking and structural analysis,five amino acid sites lining the substrate-binding pocket of P.putida KT2440 were selected for saturation mutation.The saturation mutant strains of Ser10,His89,Arg177 and Arg321 almost completely lost the enzyme activity toward L-threonine,and eight mutants of the Asp93 showed 3-8 times higher enzyme activity toward L-threonine than that of the wild type,among which Asp93His exhibited the best catalytic efficiency,and the enzyme activities toward L-threonine and L-allo-threonine were 52214 and68898 U·mg^-1,which were 9.4 times and 4.8 times higher than that of the wild type,respectively.In addition,the k_cat/K_m values of Asp93His for L-threonine and L-allo-threonine were 2925 and 4515 s^-1·m M^-1,which were 3.6 times and 6.6 times than that of the wild type,respectively.Moreover,the denaturation temperature of Asp93His was54.2°C,which was 5°C higher than that of the wild type.It was found that Asp93His shortened the distance between its side chain and the hydroxyl and amino groups of the substrate and formed a p-p bond with His133.Therefore,the mutant enzyme Asp93His showed improved catalytic efficiency and thermal stability.In the biosynthesis of 4-methylsulfonylphenylserine,it was found that the enzyme activities in the retro-aldol reaction and aldol reaction have an unequal relationship.Thus,it is necessary to establish an effective method for determining synthesis direction enzyme activity.（3）Improve the synthesis efficiency of KT2440 based on the aldol reaction activity by directed evolution.Established a 2,4-dinitrophenylhydrazine（DNPH）high-throughput screening method to detect the enzyme activity of random mutation libraries constructed by error-prone PCR technology.A total of 5 amino acid sites located near the catalytic pocket were screened out,after saturation mutation,13 variants showing improved enzyme activity in the aldol reaction were identified.Among which Ala9Val,Tyr13Lys and Tyr312Arg had the best catalytic activity in the synthesis of 4-methylsulfonylphenylserine with KT2440 and these variants as catalysts.After iterative combined mutation,A9V/Y13K/Y312R afford 72.6%conversion and 86.1%de value,representing 2.2-and 4.9-fold higher than those of wild type,respectively.The molecular catalytic mechanism analysis showed that the variant A9V/Y13K/Y312R reshaped the binding pocket and enhanced the interaction between the product and the amino acid sites lining the catalytic pocket,therefore the enzyme improved its catalytic efficiency.（4）Improve the synthesis efficiency of KT2440 by modification of product channel and cofactor binding domain.Through molecular docking and structural analysis,32 key amino acid sites in the product binding region and cofactor binding region were selected.Among them,nine potential sites related to catalytic function were determined and mutated after affinity analysis,and V-Q-Y was selected as the building block for triple code saturation mutagenesis.Three functional targets Asn12,Tyr35 and Ala176 were obtained,and 13 variants with improved enzyme activities were obtained after saturation mutation,among which the enzyme activities of Asn12Gln,Tyr35Trp and Ala176Val were increased to 13.33,12.81 and 10.64 U·mg^-1,respectively.In addition,in the iterative combination mutation,the enzyme activity of N12Q/Y35W/A176V increased to 16.32 U·mg^-1,and the k_cat/K_m value was 2.8-fold improvements relative to the wild-type.Furthermore,N12Q/Y35W/A176V showed78.3%conversion and 95.1%de value in the synthesis of 4-methylsulfonylphenylserine and exhibited improved conversion and de values for other series of benzaldehyde derivatives.Inaddition,thecombinatorialmutant A9V/N12Q/Y13K/Y35W/A176V/Y312R afford 82.6%conversion and 96.3%de value in the synthesis of 4-methylsulfonylphenylserine,representing 2.6-fold and 5.6-fold improvements relative to the wild-type.