Study On The Modification Of Catalytic Activity And Thermal Stability Of Methyl Parathion Hydrolase

Methyl parathion hydrolase（EC 3.1.8.1,MPH）belongs to theβ-lactamase family is an enzyme that can catalyze the hydrolysis of methyl parathion,ethyl parathion and other organophosphorus.Currently,MPH has been applied in the treatment of water and soil contaminated by organophosphorus pesticides,the preparation of highly sensitive organophosphorus enzyme sensors,the preparation of functional cleaning agents to remove pesticide residues,and the development of new screening marker genes.In this study,MPH from Pseudomonas sp.wbc-3 was expressed and purified in E.coli,which enzymatic properties were studied.The mechanism of improving the enzyme activity of MPH by potassium ions was studied.The catalytic efficiency and stability of MPH were improved by the strategy of site-directed mutation and small peptide fusion.The main results were listed as follows:（1）Purification and enzymatic properties of MPHThe 10×His tags were fused to the N-terminal and C-terminal of Pseudomonas sp.MPH,respectively to obtain His10-MPH and MPH-His10,which were expressed in E.coli BL21（DE3）.SDS-PAGE analysis showed that MPH-His10 could be purified to a single band（33k Da）by nickel column affinity chromatography and gel filtration chromatography.In comparison,His10-MPH also contained a band with a molecular weight of 28 k Da.The 28 k Da protein band was an incomplete degradation product of the missing C-terminal of His10-MPH identified by flight mass spectrometry.The degradation site was located in the range of G317-V327.The thermal inactivation of MPH-His10 was consistent with the first-order reversible inactivation model.Its k_d and k_r at 55°C were 0.11 min^-1 and 0.076 min^-1,respectively.These results lay a good foundation for further research on the functional-structural relationship of MPH.（2）The targets of promoting MPH enzyme activity by potassium ionsTo reduce the effect of K⁺initial position on its interaction with the enzyme,an initial dynamic conformation with a K⁺/water molecule/MPH sandwich structure and a specific K⁺aqueous environment was designed.The enrichment factor and dwell time were defined to characterize the enrichment capacity of MPH amino acid residues for potassium ions.Alanine mutation was carried out on the preferred amino acids in the sensitive region whose enrichment factors were higher than 0.1,and the amino acid targets of potassium ions acting on MPH were confirmed to be E94,D191,Q272,and N329.To improve the analysis efficiency,City Apps,which could quickly analyze the interaction between enzymes and monovalent metal ions,was written in python with Modeller and Amber as the core.City Apps provides tool support for studying the interactions between univalent metal ion interactions and enzymes.（3）Study on the catalytic activity of MPH promoted by K⁺The complex conformation of MPH with methyl-parathion and ethyl-parathion in aqueous solvent was constructed through docking and molecular dynamics simulation.Through a screening method based on free energy probability,L67,V97,F119,D151,T271,L273 were selected as the modified targets.For saturated mutations in the catalytic activity centers（19sites）,the preliminary screening results showed that 7 of the eight mutants that could significantly increase the substrate affinity or catalytic activity were distributed in L67,V97,D151,T271,and L273.Compared with the wild enzyme,the k_cat/K_m of T271S and T271V to methyl-parathion increased by 224.3%and 118.7%,respectively.Structural analysis showed that T271S formed a hydrophobic bond with the nitro group of methyl-parathion,which stabilized its conformation in the active pocket.The screening method based on free energy probability provides a new idea for semi-rational modification of enzymes.（4）Improving the stability of MPH through fusing SAP at the terminalThe N-terminal or C-terminal of MPH was fused with Self-assembling amphipathic peptide（SAP）.Compared with wild enzymes,T₅₀ and T_m of SAP3-MPH increased by6.2°C and 6.0°C respectively.SAP3-T271S was obtained by the compound mutation of SAP3-MPH and T271S,which improving catalytic efficiency.Compared with the wild type,T₅₀ and T_m of SAP3-T271S increased by 4.4?C and 5.5?C,respectively,which K_m decreased by 60.9%,and k_cat/K_m increased by 210.4%.The molecular dynamics of the small peptide monomers indicated that the small peptides SAP2,SAP3,and SAP4 had the potential of oligomerization.Hydrophobic and hydrophilic amino acids of P2 were distributed at the poles ofα-helix.There was a positive correlation between stability of MPH/SAP fusion proteins and oligomerization potential of SAP,which provides a theoretical basis for further design of SAP to improve the stability of enzyme.