In particular, elimination of heme results in a significant decrease in Tm, which is more than half as low as that of the SBP holoprotein. This strongly implies that interactions between heme and neighboring residues may contribute to the high thermodynamic stability of SBP. Based on this experimental observation, the interactions of the heme and neighboring residues of PcLiP01 were investigated by superimposing the 3D structures of SBP and PcLiP01.
Also, the synergistic effect of multiple mutants observed by the combination of single mutants led to the higher Tm of 12.1 °C rather than that of the wild type. Moreover, mutants showed similar and even higher catalytic activity compared to that of wild-type PcLiP01, strongly implying that newly introduced interactions with heme caused no significant trade-off between thermodynamic stability and activity. By this approach, the higher thermodynamic stability of heme-containing enzymes is expected without any sacrifice of activity.
Introduction
This approach strongly suggests that identification of heme-interacting protein residues will generally be the applicable method to improve the thermodynamic stability of heme-containing proteins.
Results and discussion
Mutant construction and measurement of melting temperature of PcLiP01 mutants 7
By superimposing the structure of SBP and PcLiP01, six residues (A36, E40, G86, V181, H39 and V158) of PcLiP01 were identified as they overlapped with residues interacting with heme of SBP and no characteristic interaction with heme of PcLiP01 (Fig) did not. 1 and Table 1). To determine the thermodynamic stability of mutants, melting temperature (Tm) of the wild type and mutants of PcLiP01 was measured against 0.1M Britton-Robinson buffers with variable pH (pH 2.5-6.0) based on melting curve analysis using Real-Time PCR ( Fig. S1 ). Tm of the wild type and mutants tends to decrease as pH value increases (Fig. 2).
Heme's propionyl group is believed to interact with both E40S and V181A, which could be critical for stabilizing the overall structure of PcLiP01. In our previous experiment to improve the thermodynamic stability of PcLiP01, the A55R/N156E/H239E mutant of PcLiP01 with newly formed salt bridges showed increased acid stability and thermodynamic stability [19]. Moreover, the S49C/A67C/H239E mutant of PcLiP01 with newly introduced disulfide bond and salt bridges showed an increase in Tm of 2.8 °C higher at pH 2.5 than that of the wild-type PcLiP01, by increasing the stiffness of the loop-helical structure strengthen. calcium binding sites [20].
Therefore, an increase of Tm 9.8°C for the V181A single mutant is believed to be very impressive and the interaction between heme and protein may be more critical in the thermal tolerance of PcLiP01. The Tm of wild type and mutants of PcLiP01 was measured through Differential Scanning Fluorometry using real-time PCR with 0.1 M Britton-Robinson buffer at different pH values (pH and 6.0). This means that heme can greatly contribute to the thermodynamic stability of the enzyme as a cornerstone through interactions between heme and ambient residues.
We expected synergistic effect by combining mutation sites that show higher thermodynamic stability than the wild type PcLiP01. First, according to the result of Figure 2, single mutants (H39A, E40S, V158F and V181A) that showed increased Tm than the wild type PcLiP01 were selected for multiple mutations and double mutants were constructed. This experimental result implies the synergistic effect of interactions between heme and neighboring residues and combination of mutation sites seems to play a decisive role for improving thermodynamic stability of enzyme.
This result suggests that the interaction with heme may contribute to the significant increase not only in the thermodynamic stability but also in the kinetic thermostability of the enzyme. Inactivation constant (kd) and half-life time (t1/2) of wild type and mutants of PcLiP01 measured in a 50°C incubator. Linear regression for the determination of the first-order inactivation constant (kd) of multiple kinetically thermostable mutants of PcLiP01.
Kinetic parameters of thermodynamically stable multiple mutants of PcLiP01
Kinetic parameters of the wild type and mutants of PcLiP01 were determined by the enzyme activity with different concentrations of substrate, VA and plotted by linear regression of the Hanes-Woolf plot. The correlation between kcat and half-life calculated by the study of kinetic thermostability.
Resistance of PcLiP01 mutant against oxidant
Inactivation constant (kd) and half-life (t1/2) of the wild type and the triple mutant of PcLiP01 incubated in 25 µM H2O2. In the case of wild-type PcLiP01, distortion of the overall structure was observed above 328K because beta sheets were missing and the interactions with heme were also broken (Fig. 8B). Tang, L.-P., et al., Metal and ligand effects on the stability and electronic properties of crystalline two-dimensional metal-benzene hexathiolate coordination compounds.
Ghosh, S., et al., Engineering salt bridge networks between transmembrane helices confers thermostability in G protein-coupled receptors. Missimer, J.H., et al., Configurational entropy illuminates the role of salt bridge networks in protein thermostability. Remeeva, A., et al., Effects of proline substitutions on the thermostable LOV domain from Chloroflexus aggregans.
Tanghe, M., et al., Disulfide bridges as essential elements for the thermostability of the lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor. Wang, Z., et al., Recent advances in Ni-mediated ethylene chain growth: effects of the nimine donor ligand on catalytic activity, thermal stability, and oligo-/polymeric structure. Martínez, Á.T., et al., Biodegradation of lignocellulose: microbial, chemical and enzymatic aspects of fungal attack of lignin.
Semba, Y., et al., Ancestral amino acid substitution improves the thermal stability of recombinant lignin peroxidase from the white rot fungus, Phanerochaete chrysosporium strain UAMH 3641. Son, H., et al., Extra disulfide and ionic salt bridge improve the thermostability of lignin peroxidase H8 under acidic conditions. Wang, H.-J., et al., Polarity change of a calcium site induces a hydrophobic interaction network and enhances Cel9A endoglucanase thermostability.
Land, H., et al., B-factor guided proline substitution in the amine transaminase of Chromobacterium violaceum: Evaluation of the proline rule as a method to stabilize the enzyme. Weltz, J.S., et al., Reduced enzyme dynamics after multipoint covalent immobilization leads to a trade-off between stability and activity. Malomo, S.O., et al., Suicidal inactivation of horseradish peroxidase by excess hydrogen peroxide: effects of reaction pH, buffer ion concentration, and redox mediation.
Molecular dynamics simulation
Conclusion
The current research aimed at improved thermodynamic stability of PcLiP01 through mutations 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 PcLiPO1. This research suggests a promising approach for increased thermodynamic stability of heme-containing enzymes by introducing interactions with heme as a keystone.
Materials and Methods
- Production and purification of wild type PcLiP01 and its mutants
- Specific activity of PcLiP01 and its mutants
- Determination of melting temperature (T m ) of wild type PcLiP01 and its mutants
- Catalytic properties and kinetic parameters for substrate VA
- Measurement of H 2 O 2 resistance of wild type and triple mutant
- Molecular dynamic (MD) simulation
Finally, dialyzed proteins were purified using a HiTrap Q HP column via an ÄKTA FPLC chromatography system (GE Healthcare Life Sciences) and fractions showing higher than 2.5 Reinheitszahl (RZ) values (A409/A280) were collected and stored with using a dialysis tube in 10 mM sodium acetate buffer (pH 6) with 5 mM CaCl2. The catalytic properties of the enzyme were determined with different concentrations of VA solution, and the range of VA solution was from 50 to 2000 μM. To determine the kinetic stability of the enzyme under the conditions, enzyme stocks were stored in 10 mM sodium acetate buffer (pH 6) with 5 mM CaCl2 at.
After in-silico mutation, protonation was applied at pH 3.0 with the calculations of the protein ionization and residue pKa values. Kim, In silico-engineered lignin peroxidase from Phanerochaete chrysosporium shows improved acid stability for depolymerization of lignin. Kim, Recombinant lignin peroxidase-catalyzed decolorization of melanin using in-situ generated H2O2 for application in whitening cosmetics.
