2. Results and discussion
2.6 Resistance of PcLiP01 mutant against oxidant
PcLiP01 catalyzes hydrogen peroxide-dependent oxidation as a heme-containing peroxidase. Resting state iron(III) of heme is oxidized by hydrogen peroxide as an electron acceptor to form compound I which is an oxo-iron(IV). Then, compound I is reduced by non-phenolic aromatic substrates, such as veratryl alcohol, to form cationic radicals and compound II which is an oxo-iron(IV). Finally, second substrate reduces compound II to resting ferric oxidation state and completes the catalytic cycle of PcLiP01. In the absence of non-phenolic aromatic substrates as electron donor, the addition of excessive hydrogen peroxide results in the inactive enzyme form, compound III, and this is a suicide inactivation mechanism of peroxidases[30-33]. When peroxidases are exposed to excessive hydrogen peroxide, enzymes are irreversibly damaged and inactivated. Therefore, stabilization of peroxidases against H2O2
is indispensable consideration.
Triple mutant(E40S/G86I/V181A) of PcLiP01 showing the most improved thermodynamic stability among PcLiP01 mutants was tested for the resistance to H2O2. Wild type and triple mutant(E40S/G86I/V181A) of PcLiP01 were stored in 10mM sodium acetate buffer of pH 6 with 25 µM of H2O2 without any aromatic substrates in a 25°C incubator and residual activity was measured at 0.1M Britton-Robinson buffer of pH 3 with 2mM of veratryl alcohol and 250 µM of H2O2 in room temperature (Figure 7). As shown in Table 4, the half-life time of triple mutant was about 4.7-fold longer than that of the wild type in excessive H2O2 condition. In the result, prevention of irreversible heme destruction of compound III is expected by strengthening structures through introduction of newly formed interactions between the porphyrin ring of heme and neighboring residues of PcLiP01.
.
20
Table 4. Inactivation constant(kd) and half-life time(t1/2) of the wild type and triple mutant of PcLiP01 incubated in 25 µM of H2O2
kd (min-1) t1/2 (min) Fold
WT 0.63 ×10-2 79.9 -
E40S/G86I/V181A 0.16 ×10-2 372.9 4.7
21
Time(min)
0 100 200 300 400
ln(residual activity )
-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0
WT
E40S/G86I/V181A
Figure 7. Linear regression for determining 1st order inactivation constant (kd) of triple mutants of PcLiP01 in harsh condition of excessive hydrogen peroxide
Inactivation constant(kd) was determined by plotting a linear regression with natural logarithm of residual activity and time. The wild type and triple mutant of PcLiP01 was incubated in 10mM sodium acetate buffer of pH 6 with 25 µM of H2O2 [34]
22 2.7 Molecular dynamics(MD) simulation
To investigate newly formed interactions between heme and residues of PcLiP01, molecular dynamics simulation was performed via Discovery Studio. Standard dynamics cascade and NAMD using NPT ensemble was performed for MD simulation with CHARMm force-field at various temperatures (308K, 328K, 348K and 373K).
As shown in Figure 8, protein chains are colored according to secondary structure. In the case of wild type of PcLiP01, distortion of the overall structure was observed above 328K as beta sheets were missing and the interactions with heme also broken (Fig 8B).
However, overall structure of triple mutant(E40S/G86I/V181A) was maintained at higher temperature (Fig 8D). Also, newly formed hydrogen bond and hydrophobic interaction between V181A residue of triple mutant and heme were observed and especially, hydrogen bond between V181A residue and carboxyl group of heme maintained its bonds even at high temperature, 348K (Fig 9). This result suggests that the newly formed interactions with heme can significantly affect the thermodynamic stability of heme-containing enzymes as a keystone of protein
23
Figure 8. The structures of wild type and triple mutant after NAMD
The structure of wild type of PcLiP01 at 308K(A) and at 328K(B) and the structure of triple mutant at 308K(C) and at 328K(D) are represented.
Protein is colored according to secondary structure. Helices are red, beta sheets are cyan, turns are green, and coils are white.
24
Figure 9. Newly formed interactions between heme and V181A residue of triple mutant for 3ns of MD simulation at 348K
MD simulation was performed at 348K with 3ns of NAMD simulation. Newly formed interactions are indicated as dotted line.
25
3. Conclusion
The present research aimed for enhanced thermodynamic stability of PcLiP01 through mutations that are expected to form new interactions with heme. As a result, mutants have not only improved thermodynamic stability but also much higher kinetic thermostability than the wild type of PcLiP01. In addition, trade-off between improved thermodynamic stability and catalytic properties is negligible.
Newly formed interactions between V181A residue and heme were confirmed by MD simulation. This research suggests a promising approach for enhanced thermodynamic stability of heme-containing enzymes by introducing interactions with heme as a keystone.
26
4. Materials and methods
4.1 Materials
Escherichia coli BL21(DE3) (Real Biotech Corporation, Taiwan) and pET-21b(+) expression vector (Novogene, USA) were used for protein overexpression. Isopropyl β-D-1-thiogalactopyranoside (IPTG;
I1401) for induction of gene expression was purchased from Duchefa Biochemie, Netherlands. Trizma hydrochloride (T5941), trizma base (T1503), sodium hydroxide (NaOH; S5881), urea (U5378), L- glutathione oxidized (G4376), hemin (51280), calcium chloride (CaCl2; C1016), potassium chloride (KCl; P9333), boric acid (H3BO3; B6768) were purchased from Sigma-Aldrich. Hydrogen peroxide (4104-44) was procured from Daejung Chemicals & Metals, South Korea. Acetic acid (CH3COOH;
31010-0330) was obtained from Junsei, Japan. Guanidine chloride (G0162), sodium acetate (CH3COONa; S0559), phosphoric acid (H3PO4; P1745) and veratryl alcohol (VA or 3,4- dimethoxybenzyl alcohol; V0020) were purchased from Tokyo Chemical Industry, Japan. HiTrap Q HP anion exchange column was used for protein purification which was procured from GE Healthcare Bio-Sciences, USA. Protein thermal shiftTM dye kit was purchased from Applied Biosystems, USA.
.
4.2 Production and purification of wild type PcLiP01 and its mutants.
Gene sequencing, expression, refolding and purification method were conducted as previously published reports[19, 20, 22]. The synthetic gene encoding PcLiP01 from Phanerochaete chrysosporium was conducted by the Bioneer Company (South Korea). The synthesized PcLiP01 gene sequences were cloned in ampicillin-resistant Escherichia coli expression vector pET-21b(+) via EcoRI and NdeI restriction sites. Site-directed mutagenesis was executed by using a pair of complementary primers with designed mutation sites that were synthesized by Macrogen. Resulting plasmid pET-21b(+)-PcLiP01 contains the isopropyl-β-D-1-thiogalactoside (IPTG) inducible T7 promoter. To express wild type and mutants of PcLiP01, each cell was cultured in LB medium with ampicillin (100 μg mL−1) at 37°C for 6 hours and induced at 25°C by adding IPTG at a final concentration of 1 mM at 0.6 of OD600. After induction, further incubation was conducted for 16 hours.
The cultured cells were harvested for lysis using a homogenizer (PandaPLUS 2000). The lysed cells were sonicated (at 1 s pulse on and 2 s pulse off, 20% amplitude, 2 mins) and then centrifuged (at 11000 rpm, 4°C, 10 min) for harvesting purified inclusion bodies. Then, inclusion bodies were solubilized in 8 M urea and then diluted with the refolding buffer for 16 hours at 4°C. The refolding
27
buffer is consisted of 0.1 M Tris-buffer (pH 8.5), 6 M guanidine hydrochloride, 0.71 mM L-glutathione oxidized, 1.8 mM CaCl2 and 25 μM hemin. The refolded proteins were concentrated with a concentrator (VIVAFLOW 200) and dialyzed a using dialysis tube (SnakeSkinTM Dialysis Tubing, 10K MWCO) in 100 mM sodium acetate buffer with 5 mM CaCl2 in the order of pH 6, pH 4 and pH 6, then stored 10 mM sodium acetate buffer(pH 6). Finally, dialyzed proteins were purified by using HiTrap Q HP column through ÄKTA FPLC chromatography system (GE Healthcare Life Sciences) and fractions showing higher than 2.5 of the Reinheitszahl (RZ) value (A409/A280) were collected and stored using dialysis tube in 10 mM sodium acetate buffer (pH 6) with 5 mM CaCl2.
4.3 Specific activity of PcLiP01 and its mutants
The enzymes activity was measured with 0.1M Britton-Robinson(BR) buffer of pH 3.0, 0.02 μM enzyme, 2 mM VA and the addition 250 μM H2O2 started the increase of absorbance at 310 nm using an extinction coefficient of produced veratraldehyde (ε310 nm = 9.3 mM−1 cm−1) within 1 min on spectrophotometer (UV-1650PC, Shimadzu, Japan) at room temperature[23].
4.4 Determination of melting temperature(Tm) of wild type PcLiP01 and its mutants using DSF The melting temperature (Tm) of the enzymes was determined with a protein thermal shiftTM dye kit in real-time PCR (Thermo Fisher Scientific, USA). Samples were prepared according to the user protocol (Thermo Fisher, USA). Enzymes were diluted to the average concentration range (0.5 mg/ml~1.0 mg/ml) with water and added to 0.1 M BR buffer with various pH values (pH 2.5 ~ 6.0). The solutions were mixed with protein thermal shift dye(20X). Total volume for each reaction was 20 μl and centrifuged at 1000 rpm for 1 min. The sample tubes were heated from 25 to 99°C with heating rate of 1.6°C/s in real- time PCR. Melt curve analysis were carried out using Protein Thermal Shift™ Software v1.4(Applied Biosystem, USA).
4.5 Catalytic properties and kinetic parameters for substrate VA
The catalytic properties of the enzyme were determined with varying concentrations of VA solution and the range of VA solution was from 50 to 2000 μM. The concentration of enzyme and H2O2 were fixed at 0.02 μM and 250 μM, respectively. Then, specific activity was determined with 0.1 M BR buffer with each concentration of VA. The kinetic parameters were estimated by applying the Hanes–Woolf plot using the mean values of triplicate data. To determine the kinetic stability of the enzyme under conditions, the enzyme stocks were stored in 10 mM sodium acetate buffer (pH 6) with 5 mM CaCl2 at
28
a 50°C incubator. Then, the specific activity was measured with 0.1M BR buffer of pH 3.0, 0.02 μM enzyme, 2 mM VA and 250 μM H2O2 after cooling in ice water. The data were analyzed with 1st order inactivation constant (kd) by linear regression of ln(residual activity) with incubation time(t).
4.6 Measurement of H2O2 resistance of triple mutant
The resistance against H2O2 of wild type and triple mutant (E40S/G86I/V181A) of PcLiP01 was measured by incubation in excessive hydrogen peroxide concentration. The enzyme stocks were incubated in 10 mM sodium acetate buffer (pH 6) with 5 mM CaCl2 and 25 µM of H2O2 at 25°C. Then, the residual activity was measured with 0.1M BR buffer of pH 3.0, 0.02 μM enzyme, 2 mM VA and 250 μM H2O2. The data were analyzed with 1st order inactivation constant (kd) by linear regression of ln(residual activity) with incubation time(t).
4.7 Molecular dynamic(MD) simulation
The 3D structure of wild-type PcLiP01 was obtained from Protein Data Bank(PDB:1B82) and the structure of triple mutant(E40S/G86I/V181A) was predicted using Discovery Studio Client 2021(Dassault Systems Biovia, USA). After in-silico mutation, protonation was applied at pH 3.0 with the calculations of the protein ionization and residue pKa values. Then, the wild type and triple mutant of PcLiP01 were solvated by adding water molecules and counter ions of 0.1 M NaCl with periodic boundary conditions. Standard dynamics cascade using the NPT ensemble and CHARMm force-field was used for MD simulation. Protocol of this simulation includes 1000 steps of Steepest Descent with RMS gradient tolerance of 3 and 2000 steps of Conjugate Gradient minimization, a set of 1ns heating, 10 ps equilibration and 10ps production. Then, 3 ns NAMD was performed at a specified temperature.
29
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