# Improving Catalytic Activity and Thermal Stability of Methyl-Parathion Hydrolase for Degrading the Pesticide of Methyl-Parathion

Authors: Cheng Shi; Song Liu; Guocheng Du
Journal: International Journal of Chemical Engineering
 (2022)

---

## Abstract

Pesticides are indispensable in today’s agriculture. Methyl-parathion hydrolase (MPH, E.C.3.1.8.1) could hydrolyze organophosphorus pesticides, including methyl-parathion. MPH could rehabilitate soil and water resources contaminated by organophosphorus pesticides. However, natural MPHs generally exhibited a low tolerance to high temperatures and low catalytic efficiency. In this study, we improved the catalytic efficiency toward methyl-parathion and the thermal stability of the MPH fromPseudomonas sp. WBC-3 through saturation mutagenesis and fusion with self-assembling amphipathic peptides (SAP). The experimental characterization showed that compared to the wild-type enzyme, the kcat/Km of the mutant T271S yielded by saturation mutagenesis was increased by 224.3% compared to the wild-type MPH. T50 and Tm of SAP3-MPH with an SAP fused at the N-terminus were increased by 6.2°C and 6.0°C, respectively. Compared to the wild-type enzyme, T271S fused with SAP3 at the N-terminus (SAP3-T271S) exhibited a 2.1-fold increase in kcat/Km without significantly affecting the thermal stability. The simultaneous improvement of the catalytic efficiency and thermal stability of MPH would be beneficial for its application in the degradation and detection of organophosphorus pesticides. Furthermore, our study provides a potential combination strategy for the design of the other enzyme preparations of pollutant degradation.


---

## Body

## 1. Introduction

The large-scale use of pesticides causes the pollution of water and soil resources [1, 2]. Methyl-parathion hydrolase (MPH, E.C.3.1.8.1) could catalyze the hydrolysis of methyl-parathion and other organophosphorus pesticides [3]. By converting to small molecules that are less toxic to mammals, MPH shows the application potential for remediating those soil and water contaminated by organophosphorus pesticides in addition to the use as a core component of fruit and vegetable detergents [4]. The degradation product (nitrophenol) solution is yellow in an alkaline environment and has an absorption peak at 405 nm [5]. Based on these features, MPH has been applied to construct enzyme-linked sensors for quantitatively detecting methyl-parathion content [6]. To date, MPH has been isolated from Pseudomonas sp. WBC-3 [3], Plesiomonas sp. M6 [7], Stenotrophomonas sp. SMSP-1 [8], Burkholderia Jiangsuensis MP-1 [9], etc. However, natural MPHs generally exhibited a low tolerance to high temperatures [10, 11], and it is thus necessary to improve their catalytic efficiency and stability.The thermal stability and catalytic efficiency of MPH have been modified by semirational design, directed evolution, or immobilization. As indicated by the crystal structure, MPH belongs to theβ-lactam family, many members of which have been well studied [3, 12]. Based on homologous amino acid sequence analysis, MPH from Burkholderia Jiangsuensis MP-1 was modified through a conserved residue substitution, yielding the mutant T64N with a 5.9-fold increase in kcat/Km [9]. Proline substitution within the flexible region enhanced the half-inactivation temperature (T50) of MPH from Ochrobactrum SP. M231 was increased by 2.7°C [13]. Moreover, the transcription factor DmpR that is activated by organophosphorus degradation products enabled the high-throughput detection of the MPH activity based on the signal of fluorescent protein [14]. By using this system, the mutant with a 100 times increase in kcat/Kmp-nitrophenyl diphenyl phosphate was finally isolated from the mutant library of Pseudomonas sp. strain CF600 MPH [14]. In addition, the thermal stability of Pseudomonas stutzeri ZK-5 MPH was remarkably enhanced when it is immobilized using cross-linked poly (γ-glutamic acid)/gelatin hydrogel [11]. However, the rational design based on enzyme-substrate interaction analysis was rare for MPH activity enhancement.The improvement in computing power makes full use of structural information to predict the key residues for enzymatic reactions [15]. The affinity between an enzyme and a substrate/inhibitor can be quantified using binding free energy [16, 17]. Despite the sacrifice of some precision, the method based on the master equation can rapidly assess the binding free energy [18]. The virtual amino acid mutation based on this method has been used to engineer the ω-transaminase, resulting in an 8.5 times increase in its catalytic efficiency [19]. In our previous study, we first reported the enhancement of enzyme thermal stability by fusing with self-assembling amphipathic peptides (SAP) [20]. By optimizing the SAP and linker sequence, the SAP fusion increased the half-life of polygalacturonate lyase, lipoxygenase, and L-asparaginase by 33.25-, 17.55- and 15.6-fold, respectively, compared to the corresponding wild-type enzyme [21]. To be noted, the SAP fusion generally showed little or even positive impact on the enzyme activity [20, 21]. Thus, the combination of virtual mutation and SAP fusion may be a feasible method to simultaneously improve the catalytic efficiency and thermal stability of an enzyme.In this study, the catalytic efficiency of the MPH fromPseudomonas sp. WBC-3 toward methyl-parathion was improved by modifying the critical residues, which were predicted using the virtual mutation based on binding free energy change. Then, the thermal stability of the MPH was enhanced by fusing SAP at the terminus. The mechanism for the enhanced catalytic efficiency was analyzed based on molecular dynamics.


## 2. Materials and Methods

### 2.1. Strains and Plasmids

E. coli JM109 (Novagen, Madison, WI, USA) was used for gene cloning. E. coli BL21 (DE3) (Novagen, Madison, WI, USA) and the plasmid pET-28a (+) (Novagen, USA) were used for the expression of MPH and its mutants.


### 2.2. Plasmid Construction

Each gene expressing the wild-type MPH or its mutants with a C-terminal 10×His tag was inserted into the expression plasmid pET-28a (+). In order to express the wild-type MPH with a 10×His tag, the gene of the MPH was amplified from the genome ofPseudomonas sp. WBC-3 by PCR using the primer pairs (28a)-mph-his10-F and (28a)-mph-his10-R as the sense and antisense primers, respectively. The plasmid pET-28a (+) was linearized by the primer pairs (28a)-F and (28a)-R. Then the amplified DNA expressing the wild-type MPH with a C-terminal 10×His tag was inserted into the linearized plasmid pET-28a (+) to generate the plasmid pET-28a/mph-his10, which was based on a one-step cloning method by the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The saturated mutant libraries of MPH and the combinatorial mutant D151E/T271S/L273 were accomplished using the MutanBEST Kit (Takara, Dalian, China) according to the manufacturer’s manual with the plasmid pET-28a/mph-his10 as the template. All the primer pairs used here are listed in Table S1. All the plasmids were transformed into E. coli BL21 (DE3) for protein expression.The plasmid expression MPH fused with an N-terminal SAP was yielded by linking the linearized plasmid pET-28a/mph-his10 and an SAP with homologous arms according to the ClonExpress II One Step Cloning Kit. The linearized plasmid pET-28a/mph-his10 was amplified by the primer pairs (pt)-mpd-F and (pt)-28a-R, while the SAP with homologous arms was amplified by primer pairs listed in TableS1. The homologous arms of each amplified SAP were 5′-AGAAGGAGATATACCATG-3′ and 5′-CCGACGCCGCCAACCACC-3′. The plasmid expression MPH fused with an SAP at the C-terminal was constructed by a similar flow. The difference was that the homologous arms were 5′-AGAAGGAGATATACCATG-3′ and 5′-CCGACGCCGCCAACCACC-3′, and the primer pairs to amplify pET-28a/mph-his10 were (pt)-28a-F and (pt)-mph-R (Table S1). The plasmids expressing SAP3-T271S were accomplished with the plasmid pET-28a/sap3-mph-his10 as the template through single-point mutation according to the manufacturer’s manual of the MutanBEST Kit (Takara, Dalian, China). All the primer pairs used here are listed in Table S1. All the plasmids were transformed into E. coli BL21 (DE3) for protein expression.


### 2.3. The Library Screening of Mutants

In the primary screening process, the seeds of the single-point and double-point saturated mutant libraries for MPH were cultured in a Luria-Bertani medium containing 50μg/ml kanamycin in 96-well plates. After overnight cultivation at 37°C, 1% (v/v) E. coli seed cultures were transferred into a Luria-Bertani medium containing 50 μg/ml kanamycin at 37°C for 2 h. Then protein production was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 37°C for 4 h. The cells were centrifuged and resuspended in phosphate buffer (20 mM, pH 7.0), which was repeated three times. After each resuscitation, the cells were dialyzed for 30 minutes. Then, the cells were freeze-thawed 3 times at −80°C for 1 minute and 37°C for 2 minutes. The positive mutation satisfied the condition that the special activity of the MPH mutant in the crude enzyme fluid was at least 30% higher than that of the wild-type MPH. The specific enzyme activity of the crude enzyme fluid was calculated by an approximate formula:(1)vspecial=vODmOD, MPH−mOD,blank,where vspecial is the special enzyme activity of wild-type MPH or its mutants in the crude enzyme fluid. vOD is the specific enzyme activity of unit OD of the crude enzyme fluid. mOD, MPH is the total protein concentration of unit OD of the strain expressing wild-type MPH or mutants. mOD, blank is the total protein concentration of unit OD of the strain containing the empty vectors pET-28a (+).


### 2.4. Protein Expression and Purification

In the rescreening process, a single colony of each recombinantE. coli strain obtained from the primary screening was inoculated into a Luria-Bertani medium containing kanamycin (50 μg/ml) for seed culture at 37°C overnight. Then, the seed culture broth was transferred into Terrific Broth with kanamycin (50 μg/ml) and grown at 37°C. When the optical density of the culture at 600 nm reached 0.6, the protein production was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 20°C for 14 h. After centrifugation, the cells were resuspended in phosphate buffer (20 mM, pH 7.4) and wall-broken by a high-pressure homogenizer (Union-Biotech, Shanghai, China) at 80 MPa for 30 s. The cell lysis buffer was subjected to affinity purification using the HisTrap HP column (GE Healthcare, USA), and the recombinant MPH or its mutants were eluted with 300 mM imidazole in phosphate buffer (20 mM, pH 7.4). After desalting on the HisTrap Desalting column (GE Healthcare, USA), the purified proteins were ready for the MPH activity assay. The protein expression and purification of MPH fused with an SAP were the same as above.


### 2.5. Enzyme Characterization

The dynamic thermal stabilities of MPH and MPH-SAP fusions were investigated by measuring the residual activity after incubating the enzyme solution in 100 mM Tris-HCl (pH 9). The half-temperature of heat inactivation (T50) was calculated by fitting the residual activity after incubating for 10 min at different temperatures from 20 to 80°C.The thermodynamic stability parameter of melting temperature (Tm) was performed on a Q2000 nano differential scanning calorimeter (TA, Waters, USA). MPH and MPH-SAP fusions (200 μg/ml) were equilibrated for 10 min at 0°C. The unfolding process of proteins was observed at 0–100°C with a scan rate of 2°C/min. Tm values and the enthalpy changes of the protein denaturation process were calculated by the deconvolution analysis through a non-two-state model.Apparent kinetic parameters (Km and kcat) were measured in 100 mM Tris-HCl (pH 9) using methyl-parathion ranging from 15 to 120 μM or ethyl-parathion ranging from 12 to 90 μM. All the experiments were repeated three times, and the kinetic parameters were evaluated by the nonlinear fitting of the Michaelis–Menten equation.


### 2.6. Enzyme Activity Assays

The MPH enzyme activity assay was accomplished according to a previous study with a certain modification [22]. The MPH enzyme reaction was performed at 35°C in a final reaction volume of 3.2 ml containing 100 mM Tris-HCl (pH 9), 120 μM methyl-parathion, or 90 μM ethyl-parathion, and 240 ng/ml MPH. One unit of the enzyme was defined as the amount of the MPH that catalyzed the conversion of 1 μmol methyl-parathion or ethyl-parathion per minute at 35°C. Protein concentration was measured by the BCA protein assay kit (Tiangen Biotech, China).


### 2.7. Structure Analysis

Circular dichroism (CD) and fluorescence spectroscopy analysis were done according to a previous study [20]. CD analysis was accomplished by continuously collecting the ellipticity data of enzymes in the far-UV CD band of 190–250 nm MOS-450/AF-CD-STP-A (Bio-Logic, France). Fluorescence spectroscopy was monitored at 300–400 nm and excited at 280 nm.


### 2.8. Docking Analysis

The structure of MPH (PDB: 1P9E) was prepared by Discovery Studio standard procedures, and then hydrogen atoms of histidine interacting with Zn2+ were deleted. The docking between MPH and the substrate was completed by CDOCKER of Discovery Studio (https://www.3ds.com/). The radius for pose clustering to increase the diversity of docked poses was 0.1 Å. The number of rotated ligand orientations to refine for each of the conformations was 20. Other parameters of CDOCKER were set by default.


### 2.9. Molecular Dynamics

The structure of MPH was protonated through the H++ server (https://biophysics.cs.vt.edu/) at pH 9.0. The Cd2+ of chain B was replaced by Zn2+. Molecular dynamics (MD) was accomplished by Amber [23]. A complex of MPH and methyl-parathion/ethyl-parathion was solvated in the TIP3BOX with a minimum distance of 12 Å from the edge of the box. The force field ff14SB was applied to the system. The system was minimized through 5000 iterations by the steepest descent method and 2500 iterations by the conjugate gradient method. Then the system was heated to 300 K under Langevin control and balanced. The production phase lasted 50 ns, which was sampled every 1 ps. The interaction between enzyme and substrate was analyzed by the visualization software VMD [24]. The bonding rate was the ratio of the number of bonding frames to the total number of frames in the production phase.


### 2.10. Changes in Binding Free Energy

ΔΔGbind was the difference of binding free energy (ΔGbind) between the MPH mutants and the wild type, which was calculated by Discovery Studio 2017. The force field of CHARMm Polar H was applied to the complex of MPH and substrate. The sulfur atom type of methyl-parathion and ethyl-parathion was set as “ST” which could not be identified correctly by Discovery Studio 2017. Default values were used for other parameters.


## 2.1. Strains and Plasmids

E. coli JM109 (Novagen, Madison, WI, USA) was used for gene cloning. E. coli BL21 (DE3) (Novagen, Madison, WI, USA) and the plasmid pET-28a (+) (Novagen, USA) were used for the expression of MPH and its mutants.


## 2.2. Plasmid Construction

Each gene expressing the wild-type MPH or its mutants with a C-terminal 10×His tag was inserted into the expression plasmid pET-28a (+). In order to express the wild-type MPH with a 10×His tag, the gene of the MPH was amplified from the genome ofPseudomonas sp. WBC-3 by PCR using the primer pairs (28a)-mph-his10-F and (28a)-mph-his10-R as the sense and antisense primers, respectively. The plasmid pET-28a (+) was linearized by the primer pairs (28a)-F and (28a)-R. Then the amplified DNA expressing the wild-type MPH with a C-terminal 10×His tag was inserted into the linearized plasmid pET-28a (+) to generate the plasmid pET-28a/mph-his10, which was based on a one-step cloning method by the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The saturated mutant libraries of MPH and the combinatorial mutant D151E/T271S/L273 were accomplished using the MutanBEST Kit (Takara, Dalian, China) according to the manufacturer’s manual with the plasmid pET-28a/mph-his10 as the template. All the primer pairs used here are listed in Table S1. All the plasmids were transformed into E. coli BL21 (DE3) for protein expression.The plasmid expression MPH fused with an N-terminal SAP was yielded by linking the linearized plasmid pET-28a/mph-his10 and an SAP with homologous arms according to the ClonExpress II One Step Cloning Kit. The linearized plasmid pET-28a/mph-his10 was amplified by the primer pairs (pt)-mpd-F and (pt)-28a-R, while the SAP with homologous arms was amplified by primer pairs listed in TableS1. The homologous arms of each amplified SAP were 5′-AGAAGGAGATATACCATG-3′ and 5′-CCGACGCCGCCAACCACC-3′. The plasmid expression MPH fused with an SAP at the C-terminal was constructed by a similar flow. The difference was that the homologous arms were 5′-AGAAGGAGATATACCATG-3′ and 5′-CCGACGCCGCCAACCACC-3′, and the primer pairs to amplify pET-28a/mph-his10 were (pt)-28a-F and (pt)-mph-R (Table S1). The plasmids expressing SAP3-T271S were accomplished with the plasmid pET-28a/sap3-mph-his10 as the template through single-point mutation according to the manufacturer’s manual of the MutanBEST Kit (Takara, Dalian, China). All the primer pairs used here are listed in Table S1. All the plasmids were transformed into E. coli BL21 (DE3) for protein expression.


## 2.3. The Library Screening of Mutants

In the primary screening process, the seeds of the single-point and double-point saturated mutant libraries for MPH were cultured in a Luria-Bertani medium containing 50μg/ml kanamycin in 96-well plates. After overnight cultivation at 37°C, 1% (v/v) E. coli seed cultures were transferred into a Luria-Bertani medium containing 50 μg/ml kanamycin at 37°C for 2 h. Then protein production was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 37°C for 4 h. The cells were centrifuged and resuspended in phosphate buffer (20 mM, pH 7.0), which was repeated three times. After each resuscitation, the cells were dialyzed for 30 minutes. Then, the cells were freeze-thawed 3 times at −80°C for 1 minute and 37°C for 2 minutes. The positive mutation satisfied the condition that the special activity of the MPH mutant in the crude enzyme fluid was at least 30% higher than that of the wild-type MPH. The specific enzyme activity of the crude enzyme fluid was calculated by an approximate formula:(1)vspecial=vODmOD, MPH−mOD,blank,where vspecial is the special enzyme activity of wild-type MPH or its mutants in the crude enzyme fluid. vOD is the specific enzyme activity of unit OD of the crude enzyme fluid. mOD, MPH is the total protein concentration of unit OD of the strain expressing wild-type MPH or mutants. mOD, blank is the total protein concentration of unit OD of the strain containing the empty vectors pET-28a (+).


## 2.4. Protein Expression and Purification

In the rescreening process, a single colony of each recombinantE. coli strain obtained from the primary screening was inoculated into a Luria-Bertani medium containing kanamycin (50 μg/ml) for seed culture at 37°C overnight. Then, the seed culture broth was transferred into Terrific Broth with kanamycin (50 μg/ml) and grown at 37°C. When the optical density of the culture at 600 nm reached 0.6, the protein production was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM at 20°C for 14 h. After centrifugation, the cells were resuspended in phosphate buffer (20 mM, pH 7.4) and wall-broken by a high-pressure homogenizer (Union-Biotech, Shanghai, China) at 80 MPa for 30 s. The cell lysis buffer was subjected to affinity purification using the HisTrap HP column (GE Healthcare, USA), and the recombinant MPH or its mutants were eluted with 300 mM imidazole in phosphate buffer (20 mM, pH 7.4). After desalting on the HisTrap Desalting column (GE Healthcare, USA), the purified proteins were ready for the MPH activity assay. The protein expression and purification of MPH fused with an SAP were the same as above.


## 2.5. Enzyme Characterization

The dynamic thermal stabilities of MPH and MPH-SAP fusions were investigated by measuring the residual activity after incubating the enzyme solution in 100 mM Tris-HCl (pH 9). The half-temperature of heat inactivation (T50) was calculated by fitting the residual activity after incubating for 10 min at different temperatures from 20 to 80°C.The thermodynamic stability parameter of melting temperature (Tm) was performed on a Q2000 nano differential scanning calorimeter (TA, Waters, USA). MPH and MPH-SAP fusions (200 μg/ml) were equilibrated for 10 min at 0°C. The unfolding process of proteins was observed at 0–100°C with a scan rate of 2°C/min. Tm values and the enthalpy changes of the protein denaturation process were calculated by the deconvolution analysis through a non-two-state model.Apparent kinetic parameters (Km and kcat) were measured in 100 mM Tris-HCl (pH 9) using methyl-parathion ranging from 15 to 120 μM or ethyl-parathion ranging from 12 to 90 μM. All the experiments were repeated three times, and the kinetic parameters were evaluated by the nonlinear fitting of the Michaelis–Menten equation.


## 2.6. Enzyme Activity Assays

The MPH enzyme activity assay was accomplished according to a previous study with a certain modification [22]. The MPH enzyme reaction was performed at 35°C in a final reaction volume of 3.2 ml containing 100 mM Tris-HCl (pH 9), 120 μM methyl-parathion, or 90 μM ethyl-parathion, and 240 ng/ml MPH. One unit of the enzyme was defined as the amount of the MPH that catalyzed the conversion of 1 μmol methyl-parathion or ethyl-parathion per minute at 35°C. Protein concentration was measured by the BCA protein assay kit (Tiangen Biotech, China).


## 2.7. Structure Analysis

Circular dichroism (CD) and fluorescence spectroscopy analysis were done according to a previous study [20]. CD analysis was accomplished by continuously collecting the ellipticity data of enzymes in the far-UV CD band of 190–250 nm MOS-450/AF-CD-STP-A (Bio-Logic, France). Fluorescence spectroscopy was monitored at 300–400 nm and excited at 280 nm.


## 2.8. Docking Analysis

The structure of MPH (PDB: 1P9E) was prepared by Discovery Studio standard procedures, and then hydrogen atoms of histidine interacting with Zn2+ were deleted. The docking between MPH and the substrate was completed by CDOCKER of Discovery Studio (https://www.3ds.com/). The radius for pose clustering to increase the diversity of docked poses was 0.1 Å. The number of rotated ligand orientations to refine for each of the conformations was 20. Other parameters of CDOCKER were set by default.


## 2.9. Molecular Dynamics

The structure of MPH was protonated through the H++ server (https://biophysics.cs.vt.edu/) at pH 9.0. The Cd2+ of chain B was replaced by Zn2+. Molecular dynamics (MD) was accomplished by Amber [23]. A complex of MPH and methyl-parathion/ethyl-parathion was solvated in the TIP3BOX with a minimum distance of 12 Å from the edge of the box. The force field ff14SB was applied to the system. The system was minimized through 5000 iterations by the steepest descent method and 2500 iterations by the conjugate gradient method. Then the system was heated to 300 K under Langevin control and balanced. The production phase lasted 50 ns, which was sampled every 1 ps. The interaction between enzyme and substrate was analyzed by the visualization software VMD [24]. The bonding rate was the ratio of the number of bonding frames to the total number of frames in the production phase.


## 2.10. Changes in Binding Free Energy

ΔΔGbind was the difference of binding free energy (ΔGbind) between the MPH mutants and the wild type, which was calculated by Discovery Studio 2017. The force field of CHARMm Polar H was applied to the complex of MPH and substrate. The sulfur atom type of methyl-parathion and ethyl-parathion was set as “ST” which could not be identified correctly by Discovery Studio 2017. Default values were used for other parameters.


## 3. Results and Discussion

### 3.1. Improving the Catalytic Efficiency of MPH via Site-Directed Mutations

Despite lacking substrate information, the crystal structure ofPseudomonas sp. MPH (PDB ID: 1P9E) offered the coordinates of Zn2+ that are directly involved in the catalytic reactions [12]. The docking analysis using methyl-parathion as the ligand was conducted to identify those residues critical for the MPH catalytic reaction. The hydrophobic pocket with two Zn2+ was defined as the docking sphere with a 12.0 Å radius. The 200 docked methyl-parathions were clustered according to RMSD (root mean square deviation), and 40 poses with RMSD values higher than 4 Å were eliminated (Figure S1(a)). Based on the rest of the poses, the radius of the docking sphere was reduced to 8.5 Å and used for the next round of docking analysis. Among the 200 methyl-parathion poses, the pose that exhibited the lowest CDOCKING energy was subjected to MD analysis (Figure S1(b)), yielding the final conformation of the MPH docked with methyl-parathion (Figure 1). The 20 MPH residues within 5 Å of methyl-parathion were subjected to a virtual saturation mutation using DS calculated mutation energy (binding). As shown in Figure 2, the virtual saturation mutation induced obvious changes in binding free energy. Generally, the decrease in binding free energy of the mutants indicates a higher affinity toward the substrate, while extremely high substrate affinity may cause wrong substrate binding conformation and reduced enzyme activity [25]. To obtain the variants with improved catalytic activity, we experimentally characterized those virtual saturation mutations at L67, V97, F119, D151, T271, and T273 (Figure 2). For the virtual saturation mutation at each of these sites, the minimal binding energy change was less than −0.75 kcal/mol and over 25% of the mutations had binding energy changes below 0.Figure 1
The conformation ofPseudomonas sp. WBC-3 MPH and the methyl-parathion complex. The MPH and methyl-parathion complex were constructed using CDOCKER of Discovery Studio, and the docking pose was further optimized by MD analysis. MPH and Zn2+ are shown in carton and deep purple ball, respectively. The methyl-parathion is shown in lines, while the residues within 5 Å of methyl-parathion are shown as surface in different views.Figure 2
The changes in binding free energy of MPH virtual mutants. Each selected amino acid residue of MPH was replaced with the rest 19 residues, and the resulted MPH virtual mutants were subjected to docking analysis with methyl-parathion. The change in binding free energy of the corresponding enzyme-substrate complex was calculated by Discovery Studio. Each virtual mutant at a certain position was indicated by a bold dot, which was arranged according to its ΔΔGbind value. To differentiate the positions of each mutation, the virtual mutants at the same position were connected by one dashed line.The saturation mutation at a single site of the MPH was achieved by PCR using degenerate primers (TableS1). After cyclization, the PCR products were transformed into E. coli BL21 (DE3), and the colonies were primarily screened in 96 wells according to the catalytic activity toward methyl-parathion or ethyl-parathion. All mutants of F119 were inactivated. The colonies with improved catalytic activity were cultivated in flasks, and the MPH mutants were purified using nickel affinity chromatography. Then, the purified MPH mutants were subjected to kinetic analysis using methyl-parathion or ethyl-parathion as the substrate (Table 1). When methyl-parathion was used as the substrate, L67, V97, and D151 mutants did not show enhanced kcat/Km. Finally, we identified two mutations at T271 benefiting the catalytic efficiency of MPH toward methyl-parathion. When methyl-parathion was used as the substrate, the kcat/Km values of the mutant T271S and T271V were 224.3% and 118.7% higher than that of the wild-type MPH, respectively. Compared to the wild-type enzyme, the Km values of T271S and T271V were decreased by 62.2% and 60%, respectively. These results suggested the critical role of the residue 271 in the enzyme affinity against methyl-parathion. However, these mutations have little effect on the catalytic reaction toward ethyl-parathion. In contrast to the mutations at residue 271, the two mutations at residue 273 exerted significant effects on the activities against both methyl-parathion and ethyl-parathion. L273I and L273V showed 205.6% and 198.8% increases in the catalytic efficiency toward ethyl-parathion compared to the wild-type enzyme. To be noted, both the turnover number and substrate affinity (ethyl-parathion) were enhanced by introducing the mutation L273I or L273V. However, the opposite effects on the enzyme activity were observed for L273I and L273V when methyl-parathion was used as the substrate. Thus, simultaneous saturation mutations at residues 271 and 273 were conducted to further improve the activity toward both methyl-parathion and ethyl-parathion. As shown in Table 1, we obtained only one positive mutant T271S/L273V that exhibited 142.3% and 234.8% increases in the methyl-parathion- and ethyl-parathion-hydrolysis activities, respectively. Although the activity of T271S was reduced toward methyl-parathion, the double mutation T271S/L273V slightly improved the ethyl-parathion-hydrolysis activity of L273I. These results indicated that the mutations at residues 271 and 273 showed synergies in the catalytic reaction mediated by MPH. To date, there are little reports regarding the catalytic efficiency of MPH toward methyl-parathion [9, 26]. Through the random and saturation mutations of MPH from Pseudomonas sp. WBC-3, the mutant S277Y was obtained with a 1087.8 mM−1 s−1 of kcat/Km toward methyl-parathion [27]. In the present study, the kcat/Km (methyl-parathion) of T271S reached 1173.4 mM−1 s−1, suggesting a better application prospect for the latter.Table 1
Kinetic parameters of MPH mutants.
MutantsMethyl-parathionEthyl-parathionkcat (s−1)Km (μM)kcat/Km (mM−1s−1)kcat (s−1)Km (μM)kcat/Km (mM−1s−1)WT51.9 ± 3.4143.3 ± 11.2361.8 ± 23.613.1 ± 1.8278.3 ± 14.647.1 ± 7.2L67C5.6 ± 0.695.2 ± 8.458.8 ± 7.71.6 ± 0.5681.4 ± 30.92.3 ± 0.9V97A24.4 ± 2.7100.1 ± 9.5242.4 ± 17.96.8 ± 0.8351.4 ± 44.519.4 ± 4.6D151E21.4 ± 25.4148.3 ± 46.5144.3 ± 11.28.9 ± 1.5124.2 ± 14.171.7 ± 4.3T271S63.6 ± 4.154.2 ± 8.31173.4 ± 53.915.7 ± 3.7330.6 ± 58.547.5 ± 9.7T271V45.3 ± 3.057.2 ± 4.9791.2 ± 45.413.9 ± 2.0292.5 ± 27.849.2 ± 6.3L273I13.5 ± 2.7136.4 ± 19.199.0 ± 5.332.6 ± 3.6223.5 ± 34.5145.8 ± 3.4L273V11.3 ± 1.9186.9 ± 18.360.4 ± 25.524.3 ± 7.7172.9 ± 5.2140.1 ± 13.2T271S/L273V38.5 ± 4.843.9 ± 7.1876.7 ± 67.120.3 ± 3.5128.7 ± 13.8157.7 ± 36.2SAP3-T271S62.9 ± 2.256.0 ± 3.41123.2 ± 26.412.8 ± 2.4281.5 ± 15.645.5 ± 5.2


### 3.2. Understanding the Improved Catalytic Efficiency Based on Molecular Dynamics

To understand the enhanced catalytic efficiency, the modelled structures of the T271S/methyl-parathion and L273V/ethyl-parathion complexes were built using docking and MD analysis. As shown in Figure3, ten frames of random conformation of methyl-parathion or ethyl-parathion were selected during the MD production phase. In all the frames, both T271 and L273 did not interact with methyl-parathion in the wild-type enzyme (Figure 3(a)). Although S271 did not show any interaction with the methyl-parathion, L273 formed a hydrophobic interaction with the benzene ring of the substrate in the case of T271S (Figure 3(a)). Accordingly, RMSF (root mean square fluctuation) values of the alkyl group, nitryl group, and the whole molecule of the substrate in T271S were, respectively, lower than those in the wild-type enzyme (Figure S2). To be noted, the docked methyl-parathion was close to D151 and the water molecule that were predicted to mediate the nucleophilic attack on the phosphorus center of the substrate during the catalytic process [12, 28]. The stable orientation of the substrate may be more suitable for the catalysis mediated by D151 in contrast to the less stable one. It should be noted that the Km value of T271S was reduced compared to the wild-type enzyme. Therefore, the mutation at T271 changed the orientation of the neighboring L273, inducing an enhanced enzyme affinity toward methyl-parathion via the hydrophobic interaction (Table 1). In the case of the complexes with ethyl-parathion, both wild-type enzyme and L273V formed a hydrophobic bond between residue 273 and the substrate (Figure 3(b)). However, the formation rate of the hydrophobic interaction in the former was 32.5% higher than that in the latter, suggesting that the interaction between residue 273 and the substrate was unfavorable for the orientation and degradation of ethyl-parathion. It has been reported that the MPH showed an increase in activity toward p-nitrophenyl diphenyl phosphate after replacing bulky L273 with smaller alanine [14]. Considering the smaller size of leucine than that of valine, the smaller steric hindrance at 273 in L273V may account for its enhanced activity toward ethyl-parathion compared to the wild-type enzyme. These mechanisms could also be used to explain that the MPH activity toward ethyl-parathion was higher than that against ethyl-parathion (Table 1). In addition to the relatively smaller size than ethyl-parathion, methyl-parathion did not show any interaction with L273 (Figure 3).Figure 3
The complex conformation difference between MPH and its mutant. (a) The conformation of the enzymes with methyl-parathion. (b) The conformation of the enzymes with ethyl-parathion. The enzyme-substrate complex was constructed using CDOCKER of Discovery Studio, and the docking pose was further optimized by MD analysis. The mean conformation of MPH was shown in carton. During the MD analysis, ten random samples of each substrate were selected and presented in lines. The hydrophobic bonds between enzyme and substrate were indicated by dashed lines. The active residue D151 was marked in yellow. The oxygen atoms of water and Zn2+ were shown as the red and deep purple balls, respectively.
(a)(b)


### 3.3. Improving the Stability of MPH via the Terminal Fusion with SAP

Our previous results have indicated that fusing with SAP at the terminus could improve the enzyme thermal stability [20, 21]. To improve the thermal stability of MPH, an SAP was fused to the N- or C-terminus of MPH via a PT-Linker, and a tag with 10 histidine residues was fused at the C-terminus for affinity purification (Figure 4). Six SAPs were tested for enhanced thermal stability (Table S2). MPH-SAP fusions were expressed intracellularly and purified after cell disruption. The T50 (a kinetic stability parameter that expresses the ability of an enzyme to maintain the reaction efficiency at a specific time) and Tm (a thermodynamic stability parameter that concerns the ability of an enzyme to resist the unfolding process) were used to characterize the thermal stability. Among the tested SAPs, SAP2, SAP3, and SAP4 improved the thermal stability of MPH, and fusing SAPs at the N-terminus of MPH was generally more efficient than fusion at its C-terminus (Table 2). SAP3 fused at the N-terminus of MPH (SAP3-MPH) achieved the highest thermal stability, with 6.2°C and 6.0°C increases in T50 and Tm than the wild-type enzyme, respectively (Table 2). However, both terminus fusion protein (SAP3-MPH-SAP3) lost its catalytic activity. The crystal structure shows that the N-terminal amino acid is no more than 5 Å away from the C-terminal amino acid of the other monomer [3], which may result in the inability to accommodate two SAPs near the end of MPH. To simultaneously improve the catalytic efficiency and stability, we fused the SAP to the N-terminus of T271S, yielding SAP3-T271S. Although less stable than SAP3-MPH, SAP3-T271S exhibited 4.4°C and 5.5°C increases in T50 and Tm than the wild type, respectively (Table 2). Compared with T271S, SAP3-T271S made up for the loss of stability caused by site-directed mutation (Table 2). To be noted, the SAP fusion has little impact on the activity of T271S toward methyl-parathion or ethyl-parathion (Table 2). When methyl-parathion was used as the substrate, kcat/Km of SAP-T271S was 2.1-fold higher than that of the wild-type enzyme (Table 1). As T271S did, SAP-T271S shared a similar catalytic efficiency against ethyl-parathion with the wild type.Figure 4
Expression cassettes of MPH fused with SAP. (a) SAP fused at the N-terminus of MPH via a PT-linker (PTPPTTPTPPTTPTPTP). (b) SAP fused at the C-terminus of MPH via a PT-linker. All the expression cassettes were fused with a tag composed of ten histidine residues. Six SAPs were totally used in the constructs (TableS2).
(a)(b)Table 2
Thermal stability parameters of MPH fused with SAP.
Fusion enzymeT50 (°C)Tm (°C)WT55.162.4T271S54.862.2SAP1-MPH55.562.4SAP2-MPH58.266.5SAP3-MPH61.368.4SAP4-MPH57.766.5SAP5-MPH53.760.6SAP6-MPH54.961.3SAP3-T271S59.567.9MPH-SAP155.962.4MPH-SAP257.263.7MPH-SAP359.466.8MPH-SAP456.764.0MPH-SAP554.661.3MPH-SAP654.362.2Secondary and tertiary structure analyses of the MPH and its SAP fusions were conducted using CD (Figure5(a)) and fluorescence spectroscopy (Figure 5(b)), respectively. However, no significant difference was observed in secondary and tertiary structure among the wild type, SAP3-MPH, and SAP3-T271S. Generally, SAP fusion was predicted to facilitate the enzyme oligomerization through intermolecular hydrophobic interactions [20, 21], which showed little impact on the secondary and tertiary structure of the enzyme [20]. Therefore, SAP3-MPH and SAP3-T271S may also adopt the same stabilization. To understand why different SAPs caused different effects on MPH, the structure of SAPs was simulated by MD (Figure S3). According to the distribution of hydrophobic and hydrophilic amino acids of SAP, the possible patterns of oligomer formation mediated by different SAPs were deduced (Figure 6). SAP1, SAP5, and SAP6 were difficult to oligomerize. SAP3 tends to form the larger scale oligomer, which provides a possible explanation for the higher stability of SAP3-MPH.Figure 5
The effect of SAP fusion on the structure of the MPH mutants. (a) CD spectrometry. (b) Steady-state fluorescence spectra.
(a)(b)Figure 6
The patterns of oligomers mediated by different SAPs. The analysis of hydrophobic and hydrophilic amino acids shows the potential patterns of oligomers mediated by SAP2 (a), SAP3 (b), and SAP4 (c). The hydrophobic and hydrophilic regions are shown in brown and green, respectively.
(a)(b)(c)In previous studies, theT50 value of the MPH from Ochrobactrum sp. M231 was enhanced through the residue replacement with rigid proline [13] and the introduction of surface ionic bonds [29], achieving the mutant P76D/P78K with 68°C of T50. However, the kcat/Km of P76D/P78K against methyl-parathion was just 178.33 mM−1s−1 [29], less than one-fifth of that of SAP-T271S. Despite being less stable than the reported mutant, SAP-T271S still showed a catalytic efficiency advantage.


## 3.1. Improving the Catalytic Efficiency of MPH via Site-Directed Mutations

Despite lacking substrate information, the crystal structure ofPseudomonas sp. MPH (PDB ID: 1P9E) offered the coordinates of Zn2+ that are directly involved in the catalytic reactions [12]. The docking analysis using methyl-parathion as the ligand was conducted to identify those residues critical for the MPH catalytic reaction. The hydrophobic pocket with two Zn2+ was defined as the docking sphere with a 12.0 Å radius. The 200 docked methyl-parathions were clustered according to RMSD (root mean square deviation), and 40 poses with RMSD values higher than 4 Å were eliminated (Figure S1(a)). Based on the rest of the poses, the radius of the docking sphere was reduced to 8.5 Å and used for the next round of docking analysis. Among the 200 methyl-parathion poses, the pose that exhibited the lowest CDOCKING energy was subjected to MD analysis (Figure S1(b)), yielding the final conformation of the MPH docked with methyl-parathion (Figure 1). The 20 MPH residues within 5 Å of methyl-parathion were subjected to a virtual saturation mutation using DS calculated mutation energy (binding). As shown in Figure 2, the virtual saturation mutation induced obvious changes in binding free energy. Generally, the decrease in binding free energy of the mutants indicates a higher affinity toward the substrate, while extremely high substrate affinity may cause wrong substrate binding conformation and reduced enzyme activity [25]. To obtain the variants with improved catalytic activity, we experimentally characterized those virtual saturation mutations at L67, V97, F119, D151, T271, and T273 (Figure 2). For the virtual saturation mutation at each of these sites, the minimal binding energy change was less than −0.75 kcal/mol and over 25% of the mutations had binding energy changes below 0.Figure 1
The conformation ofPseudomonas sp. WBC-3 MPH and the methyl-parathion complex. The MPH and methyl-parathion complex were constructed using CDOCKER of Discovery Studio, and the docking pose was further optimized by MD analysis. MPH and Zn2+ are shown in carton and deep purple ball, respectively. The methyl-parathion is shown in lines, while the residues within 5 Å of methyl-parathion are shown as surface in different views.Figure 2
The changes in binding free energy of MPH virtual mutants. Each selected amino acid residue of MPH was replaced with the rest 19 residues, and the resulted MPH virtual mutants were subjected to docking analysis with methyl-parathion. The change in binding free energy of the corresponding enzyme-substrate complex was calculated by Discovery Studio. Each virtual mutant at a certain position was indicated by a bold dot, which was arranged according to its ΔΔGbind value. To differentiate the positions of each mutation, the virtual mutants at the same position were connected by one dashed line.The saturation mutation at a single site of the MPH was achieved by PCR using degenerate primers (TableS1). After cyclization, the PCR products were transformed into E. coli BL21 (DE3), and the colonies were primarily screened in 96 wells according to the catalytic activity toward methyl-parathion or ethyl-parathion. All mutants of F119 were inactivated. The colonies with improved catalytic activity were cultivated in flasks, and the MPH mutants were purified using nickel affinity chromatography. Then, the purified MPH mutants were subjected to kinetic analysis using methyl-parathion or ethyl-parathion as the substrate (Table 1). When methyl-parathion was used as the substrate, L67, V97, and D151 mutants did not show enhanced kcat/Km. Finally, we identified two mutations at T271 benefiting the catalytic efficiency of MPH toward methyl-parathion. When methyl-parathion was used as the substrate, the kcat/Km values of the mutant T271S and T271V were 224.3% and 118.7% higher than that of the wild-type MPH, respectively. Compared to the wild-type enzyme, the Km values of T271S and T271V were decreased by 62.2% and 60%, respectively. These results suggested the critical role of the residue 271 in the enzyme affinity against methyl-parathion. However, these mutations have little effect on the catalytic reaction toward ethyl-parathion. In contrast to the mutations at residue 271, the two mutations at residue 273 exerted significant effects on the activities against both methyl-parathion and ethyl-parathion. L273I and L273V showed 205.6% and 198.8% increases in the catalytic efficiency toward ethyl-parathion compared to the wild-type enzyme. To be noted, both the turnover number and substrate affinity (ethyl-parathion) were enhanced by introducing the mutation L273I or L273V. However, the opposite effects on the enzyme activity were observed for L273I and L273V when methyl-parathion was used as the substrate. Thus, simultaneous saturation mutations at residues 271 and 273 were conducted to further improve the activity toward both methyl-parathion and ethyl-parathion. As shown in Table 1, we obtained only one positive mutant T271S/L273V that exhibited 142.3% and 234.8% increases in the methyl-parathion- and ethyl-parathion-hydrolysis activities, respectively. Although the activity of T271S was reduced toward methyl-parathion, the double mutation T271S/L273V slightly improved the ethyl-parathion-hydrolysis activity of L273I. These results indicated that the mutations at residues 271 and 273 showed synergies in the catalytic reaction mediated by MPH. To date, there are little reports regarding the catalytic efficiency of MPH toward methyl-parathion [9, 26]. Through the random and saturation mutations of MPH from Pseudomonas sp. WBC-3, the mutant S277Y was obtained with a 1087.8 mM−1 s−1 of kcat/Km toward methyl-parathion [27]. In the present study, the kcat/Km (methyl-parathion) of T271S reached 1173.4 mM−1 s−1, suggesting a better application prospect for the latter.Table 1
Kinetic parameters of MPH mutants.
MutantsMethyl-parathionEthyl-parathionkcat (s−1)Km (μM)kcat/Km (mM−1s−1)kcat (s−1)Km (μM)kcat/Km (mM−1s−1)WT51.9 ± 3.4143.3 ± 11.2361.8 ± 23.613.1 ± 1.8278.3 ± 14.647.1 ± 7.2L67C5.6 ± 0.695.2 ± 8.458.8 ± 7.71.6 ± 0.5681.4 ± 30.92.3 ± 0.9V97A24.4 ± 2.7100.1 ± 9.5242.4 ± 17.96.8 ± 0.8351.4 ± 44.519.4 ± 4.6D151E21.4 ± 25.4148.3 ± 46.5144.3 ± 11.28.9 ± 1.5124.2 ± 14.171.7 ± 4.3T271S63.6 ± 4.154.2 ± 8.31173.4 ± 53.915.7 ± 3.7330.6 ± 58.547.5 ± 9.7T271V45.3 ± 3.057.2 ± 4.9791.2 ± 45.413.9 ± 2.0292.5 ± 27.849.2 ± 6.3L273I13.5 ± 2.7136.4 ± 19.199.0 ± 5.332.6 ± 3.6223.5 ± 34.5145.8 ± 3.4L273V11.3 ± 1.9186.9 ± 18.360.4 ± 25.524.3 ± 7.7172.9 ± 5.2140.1 ± 13.2T271S/L273V38.5 ± 4.843.9 ± 7.1876.7 ± 67.120.3 ± 3.5128.7 ± 13.8157.7 ± 36.2SAP3-T271S62.9 ± 2.256.0 ± 3.41123.2 ± 26.412.8 ± 2.4281.5 ± 15.645.5 ± 5.2


## 3.2. Understanding the Improved Catalytic Efficiency Based on Molecular Dynamics

To understand the enhanced catalytic efficiency, the modelled structures of the T271S/methyl-parathion and L273V/ethyl-parathion complexes were built using docking and MD analysis. As shown in Figure3, ten frames of random conformation of methyl-parathion or ethyl-parathion were selected during the MD production phase. In all the frames, both T271 and L273 did not interact with methyl-parathion in the wild-type enzyme (Figure 3(a)). Although S271 did not show any interaction with the methyl-parathion, L273 formed a hydrophobic interaction with the benzene ring of the substrate in the case of T271S (Figure 3(a)). Accordingly, RMSF (root mean square fluctuation) values of the alkyl group, nitryl group, and the whole molecule of the substrate in T271S were, respectively, lower than those in the wild-type enzyme (Figure S2). To be noted, the docked methyl-parathion was close to D151 and the water molecule that were predicted to mediate the nucleophilic attack on the phosphorus center of the substrate during the catalytic process [12, 28]. The stable orientation of the substrate may be more suitable for the catalysis mediated by D151 in contrast to the less stable one. It should be noted that the Km value of T271S was reduced compared to the wild-type enzyme. Therefore, the mutation at T271 changed the orientation of the neighboring L273, inducing an enhanced enzyme affinity toward methyl-parathion via the hydrophobic interaction (Table 1). In the case of the complexes with ethyl-parathion, both wild-type enzyme and L273V formed a hydrophobic bond between residue 273 and the substrate (Figure 3(b)). However, the formation rate of the hydrophobic interaction in the former was 32.5% higher than that in the latter, suggesting that the interaction between residue 273 and the substrate was unfavorable for the orientation and degradation of ethyl-parathion. It has been reported that the MPH showed an increase in activity toward p-nitrophenyl diphenyl phosphate after replacing bulky L273 with smaller alanine [14]. Considering the smaller size of leucine than that of valine, the smaller steric hindrance at 273 in L273V may account for its enhanced activity toward ethyl-parathion compared to the wild-type enzyme. These mechanisms could also be used to explain that the MPH activity toward ethyl-parathion was higher than that against ethyl-parathion (Table 1). In addition to the relatively smaller size than ethyl-parathion, methyl-parathion did not show any interaction with L273 (Figure 3).Figure 3
The complex conformation difference between MPH and its mutant. (a) The conformation of the enzymes with methyl-parathion. (b) The conformation of the enzymes with ethyl-parathion. The enzyme-substrate complex was constructed using CDOCKER of Discovery Studio, and the docking pose was further optimized by MD analysis. The mean conformation of MPH was shown in carton. During the MD analysis, ten random samples of each substrate were selected and presented in lines. The hydrophobic bonds between enzyme and substrate were indicated by dashed lines. The active residue D151 was marked in yellow. The oxygen atoms of water and Zn2+ were shown as the red and deep purple balls, respectively.
(a)(b)


## 3.3. Improving the Stability of MPH via the Terminal Fusion with SAP

Our previous results have indicated that fusing with SAP at the terminus could improve the enzyme thermal stability [20, 21]. To improve the thermal stability of MPH, an SAP was fused to the N- or C-terminus of MPH via a PT-Linker, and a tag with 10 histidine residues was fused at the C-terminus for affinity purification (Figure 4). Six SAPs were tested for enhanced thermal stability (Table S2). MPH-SAP fusions were expressed intracellularly and purified after cell disruption. The T50 (a kinetic stability parameter that expresses the ability of an enzyme to maintain the reaction efficiency at a specific time) and Tm (a thermodynamic stability parameter that concerns the ability of an enzyme to resist the unfolding process) were used to characterize the thermal stability. Among the tested SAPs, SAP2, SAP3, and SAP4 improved the thermal stability of MPH, and fusing SAPs at the N-terminus of MPH was generally more efficient than fusion at its C-terminus (Table 2). SAP3 fused at the N-terminus of MPH (SAP3-MPH) achieved the highest thermal stability, with 6.2°C and 6.0°C increases in T50 and Tm than the wild-type enzyme, respectively (Table 2). However, both terminus fusion protein (SAP3-MPH-SAP3) lost its catalytic activity. The crystal structure shows that the N-terminal amino acid is no more than 5 Å away from the C-terminal amino acid of the other monomer [3], which may result in the inability to accommodate two SAPs near the end of MPH. To simultaneously improve the catalytic efficiency and stability, we fused the SAP to the N-terminus of T271S, yielding SAP3-T271S. Although less stable than SAP3-MPH, SAP3-T271S exhibited 4.4°C and 5.5°C increases in T50 and Tm than the wild type, respectively (Table 2). Compared with T271S, SAP3-T271S made up for the loss of stability caused by site-directed mutation (Table 2). To be noted, the SAP fusion has little impact on the activity of T271S toward methyl-parathion or ethyl-parathion (Table 2). When methyl-parathion was used as the substrate, kcat/Km of SAP-T271S was 2.1-fold higher than that of the wild-type enzyme (Table 1). As T271S did, SAP-T271S shared a similar catalytic efficiency against ethyl-parathion with the wild type.Figure 4
Expression cassettes of MPH fused with SAP. (a) SAP fused at the N-terminus of MPH via a PT-linker (PTPPTTPTPPTTPTPTP). (b) SAP fused at the C-terminus of MPH via a PT-linker. All the expression cassettes were fused with a tag composed of ten histidine residues. Six SAPs were totally used in the constructs (TableS2).
(a)(b)Table 2
Thermal stability parameters of MPH fused with SAP.
Fusion enzymeT50 (°C)Tm (°C)WT55.162.4T271S54.862.2SAP1-MPH55.562.4SAP2-MPH58.266.5SAP3-MPH61.368.4SAP4-MPH57.766.5SAP5-MPH53.760.6SAP6-MPH54.961.3SAP3-T271S59.567.9MPH-SAP155.962.4MPH-SAP257.263.7MPH-SAP359.466.8MPH-SAP456.764.0MPH-SAP554.661.3MPH-SAP654.362.2Secondary and tertiary structure analyses of the MPH and its SAP fusions were conducted using CD (Figure5(a)) and fluorescence spectroscopy (Figure 5(b)), respectively. However, no significant difference was observed in secondary and tertiary structure among the wild type, SAP3-MPH, and SAP3-T271S. Generally, SAP fusion was predicted to facilitate the enzyme oligomerization through intermolecular hydrophobic interactions [20, 21], which showed little impact on the secondary and tertiary structure of the enzyme [20]. Therefore, SAP3-MPH and SAP3-T271S may also adopt the same stabilization. To understand why different SAPs caused different effects on MPH, the structure of SAPs was simulated by MD (Figure S3). According to the distribution of hydrophobic and hydrophilic amino acids of SAP, the possible patterns of oligomer formation mediated by different SAPs were deduced (Figure 6). SAP1, SAP5, and SAP6 were difficult to oligomerize. SAP3 tends to form the larger scale oligomer, which provides a possible explanation for the higher stability of SAP3-MPH.Figure 5
The effect of SAP fusion on the structure of the MPH mutants. (a) CD spectrometry. (b) Steady-state fluorescence spectra.
(a)(b)Figure 6
The patterns of oligomers mediated by different SAPs. The analysis of hydrophobic and hydrophilic amino acids shows the potential patterns of oligomers mediated by SAP2 (a), SAP3 (b), and SAP4 (c). The hydrophobic and hydrophilic regions are shown in brown and green, respectively.
(a)(b)(c)In previous studies, theT50 value of the MPH from Ochrobactrum sp. M231 was enhanced through the residue replacement with rigid proline [13] and the introduction of surface ionic bonds [29], achieving the mutant P76D/P78K with 68°C of T50. However, the kcat/Km of P76D/P78K against methyl-parathion was just 178.33 mM−1s−1 [29], less than one-fifth of that of SAP-T271S. Despite being less stable than the reported mutant, SAP-T271S still showed a catalytic efficiency advantage.


## 4. Conclusion

In this study, we improved the catalytic efficiency and thermal stability of the MPH fromPseudomonas sp. WBC-3 using site-directed mutation and SAP fusion. Compared to the wild-type enzyme, the resulted MPH mutant SAP-T271S exhibited a 2.1-fold increase in catalytic efficiency, and it also showed 4.4°C and 5.5°C increases in T50 and Tm, respectively. With the improved catalytic properties, SAP-T271S could be a robust candidate for the application in the degradation and detection of organophosphorus pesticides.



---

*Source: 7355170-2022-04-06.xml*