2.2.4. Effect of Magnesium (Mg2+) on MTase Activity

Although divalent cations are crucial for the activity of many viral enzymes, no study has been observed the role of divalent cations on MTase activity. Hence, MTase activity in response to varying concentrations of Mg2+ was studied. This resulted in the linear increase in MTase activity for up to 2 mM of magnesium, after which it became saturated (Figure 5A). The effect of another important cation, Ca2+, was checked for its activity of the enzyme, but no effect on MTase activity was seen (data not shown). To further validate the impact of Mg2+, the reaction was performed in the presence of EDTA, which is supposed to be a chelating agent for the magnesium. When increasing the concentration of EDTA from 0 to 8 mM, the enzyme activity decreased significantly, establishing that the presence of magnesium is essential for the MTase activity (Figure 5B). *Molecules* **2022**, *27*, x FOR PEER REVIEW 7 of 18

**Figure 5. Effect of magnesium on MTase activity**: (**A**) The graph represents MTase activity when increasing the concentration of MgCl2 up to 10 mM. (**B**) The effect of EDTA on the enzyme activity. The graphs represent the mean value of three different readings and the error bars indicate the standard deviation. **Figure 5. Effect of magnesium on MTase activity**: (**A**) The graph represents MTase activity when increasing the concentration of MgCl<sup>2</sup> up to 10 mM. (**B**) The effect of EDTA on the enzyme activity. The graphs represent the mean value of three different readings and the error bars indicate the standard deviation.

#### *2.3. Circular Dichroism (CD) Analysis 2.3. Circular Dichroism (CD) Analysis*

Chen et al. [24].

12.0 at 25 °C.

Far-UV CD experiments were performed to investigate the changes in secondary structural content of MTase at different pH values or Mg2+ concentrations [23]. The obtained MTase spectra showed two negative peaks around 208 nm and 222 nm. A positive peak at 195 nm (not shown in the figure due to high HT values at this region) reflects a characteristic α-helical spectrum of MTase (Figure 6). In addition, the negative peak at 218 indicates the presence of the β-sheets. Overall, at pH 8.0, it contains 27% α-helix, 23% βsheet, and the rest of the content as random coils, loops, turns, etc. Hence, we report that 50% of the MTase protein is comprised of α-helical and β-sheet structures. Similar percentage of α-helices and β-sheets for MTase were obtained by secondary structure prediction methods (data not shown). The secondary structure of MTase was calculated using online DichroWeb software (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) (accessed on 27 December 2021) based on the K2D model and analysed by the method of Far-UV CD experiments were performed to investigate the changes in secondary structural content of MTase at different pH values or Mg2+ concentrations [23]. The obtained MTase spectra showed two negative peaks around 208 nm and 222 nm. A positive peak at 195 nm (not shown in the figure due to high HT values at this region) reflects a characteristic α-helical spectrum of MTase (Figure 6). In addition, the negative peak at 218 indicates the presence of the β-sheets. Overall, at pH 8.0, it contains 27% α-helix, 23% β-sheet, and the rest of the content as random coils, loops, turns, etc. Hence, we report that 50% of the MTase protein is comprised of α-helical and β-sheet structures. Similar percentage of α-helices and β-sheets for MTase were obtained by secondary structure prediction methods (data not shown). The secondary structure of MTase was calculated using online DichroWeb software (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) (accessed on 27 December 2021) based on the K2D model and analysed by the method of Chen et al. [24].

**Figure 6. Far-UV CD spectra of MTase:** To determine the change in the secondary structure of MTase in the presence of N-lauryl sarcosine sodium salt and NaCl at pH 4.0, 6.0, 7.0, 8.0, 10.0, and **3.5**

**4.0**

**4.5**

**Log[SAH]** 

**5.0**

**5.5**

standard deviation.

**0.1560 0.3125 0.625 1.25 2.5 5 10**

**[MgCl2] (in mM)**

**A B**

Chen et al. [24].

*2.3. Circular Dichroism (CD) Analysis* 

**Figure 5. Effect of magnesium on MTase activity**: (**A**) The graph represents MTase activity when increasing the concentration of MgCl2 up to 10 mM. (**B**) The effect of EDTA on the enzyme activity. The graphs represent the mean value of three different readings and the error bars indicate the

**3.5**

**4.0**

**4.5**

**Log[SAH]**

**5.0**

**5.5**

**0 0.5 1 2 4 6 8**

**[EDTA] in mM**

Far-UV CD experiments were performed to investigate the changes in secondary structural content of MTase at different pH values or Mg2+ concentrations [23]. The obtained MTase spectra showed two negative peaks around 208 nm and 222 nm. A positive peak at 195 nm (not shown in the figure due to high HT values at this region) reflects a characteristic α-helical spectrum of MTase (Figure 6). In addition, the negative peak at 218 indicates the presence of the β-sheets. Overall, at pH 8.0, it contains 27% α-helix, 23% βsheet, and the rest of the content as random coils, loops, turns, etc. Hence, we report that 50% of the MTase protein is comprised of α-helical and β-sheet structures. Similar percentage of α-helices and β-sheets for MTase were obtained by secondary structure prediction methods (data not shown). The secondary structure of MTase was calculated using online DichroWeb software (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) (accessed on 27 December 2021) based on the K2D model and analysed by the method of

12.0 at 25 °C. *2.4. Structural and Thermal Stability of MTase in the Presence of Mg2+*

The structural stability of MTase was investigated in the presence of Mg2+ at different pH conditions. The experimental results revealed a minimum change in the secondary structure of MTase in the presence of Mg2+ at pH 4.0 and pH 8.0 compared to pH 6.0, pH 7.0, pH 10.0, and pH 12.0 (Figure 7). As per our observations, Mg2+ is responsible for the change in the secondary structure at different pH conditions. However, it has been found that shifting the pH values towards acidic (pH 4.0) or alkaline (pH 12.0) is responsible for the increment in the α-helix content of MTase. This observation confirmed that the MTase secondary structure in the presence of 0.2 mM Mg2+ is more stable at pH 4.0 and pH 8.0 compared to other pH values. Therefore, we selected pH 8.0 for further studies, which is closer to physiological pH. This will help provide a comparative analysis for protein activity and drug discovery. *2.4. Structural and Thermal Stability of MTase in the Presence of Mg2+* The structural stability of MTase was investigated in the presence of Mg2+ at different pH conditions. The experimental results revealed a minimum change in the secondary structure of MTase in the presence of Mg2+ at pH 4.0 and pH 8.0 compared to pH 6.0, pH 7.0, pH 10.0, and pH 12.0 (Figure 7). As per our observations, Mg2+ is responsible for the change in the secondary structure at different pH conditions. However, it has been found that shifting the pH values towards acidic (pH 4.0) or alkaline (pH 12.0) is responsible for the increment in the α-helix content of MTase. This observation confirmed that the MTase secondary structure in the presence of 0.2 mM Mg2+ is more stable at pH 4.0 and pH 8.0 compared to other pH values. Therefore, we selected pH 8.0 for further studies, which is closer to physiological pH. This will help provide a comparative analysis for protein activity and drug discovery.

**Figure 7. Far-UV CD spectra of MTase:** CD spectra at (**A**) pH 4.0, (**B**) pH 6.0, (**C**) pH 7.0, (**D**) pH 8.0, (**E**) pH 10.0, and (**F**) pH 12.0 under standard temperature in the absence and presence of 0.2 mM Mg2+; all respective buffer solutions contain N-lauryl sarcosine sodium salt and NaCl to provide structural stability at respective pH. Further, we have determined the effect of Mg2+ on MTase secondary structural **Figure 7. Far-UV CD spectra of MTase:** CD spectra at (**A**) pH 4.0, (**B**) pH 6.0, (**C**) pH 7.0, (**D**) pH 8.0, (**E**) pH 10.0, and (**F**) pH 12.0 under standard temperature in the absence and presence of 0.2 mM Mg2+; all respective buffer solutions contain N-lauryl sarcosine sodium salt and NaCl to provide structural stability at respective pH.

changes by varying the Mg2+ concentrations (Figure 8A). We found that 0.2 mM Mg2+ effectively changed the secondary structure of MTase compared to 1.0 mM of Mg2+. Furthermore, the far-UV CD spectra revealed that lower Mg2+ concentrations induced the formation of β-sheets that was proportionally not supported at higher concentrations. Moreover, we determined the thermal stability at similar conditions. The obtained experimental denaturation graphs (Figure 8B) at 0.2 mM Mg2+ showed that the metal ions effec-

Further, we have determined the effect of Mg2+ on MTase secondary structural changes by varying the Mg2+ concentrations (Figure 8A). We found that 0.2 mM Mg2+ effectively changed the secondary structure of MTase compared to 1.0 mM of Mg2+. Furthermore, the far-UV CD spectra revealed that lower Mg2+ concentrations induced the formation of β-sheets that was proportionally not supported at higher concentrations. Moreover, we determined the thermal stability at similar conditions. The obtained experimental denaturation graphs (Figure 8B) at 0.2 mM Mg2+ showed that the metal ions effectively induced the thermal stability of MTase compared to 1.0 mM (Table 1). Far UV-CD spectra and enzymatic assays also supported these results. *Molecules* **2022**, *27*, x FOR PEER REVIEW 9 of 18

**Figure 8. Far-UV CD spectra and thermal denaturation of MTase:** (**A**) Far-UV Cd spectra at standard temperature (25 °C) (**B**). Thermal denaturation spectra of MTase at 222 nm in absence and presence of 0.2 mM and 1.0 mM Mg2+. Tris buffer of pH 8.0 containing N-lauryl sarcosine sodium salt and NaCl was used in each experimental sample under minimum concentrations. **Figure 8. Far-UV CD spectra and thermal denaturation of MTase:** (**A**) Far-UV Cd spectra at standard temperature (25 ◦C). (**B**) Thermal denaturation spectra of MTase at 222 nm in absence and presence of 0.2 mM and 1.0 mM Mg2+. Tris buffer of pH 8.0 containing N-lauryl sarcosine sodium salt and NaCl was used in each experimental sample under minimum concentrations.

MTase in the presence and absence of Mg2+. This revealed changes in the structural conformational and stability of MTase at pH 8.0. **Sample System % α-Helices % β-Sheets % Random Coils and Table 1.** Percentage change in secondary structure content and thermal denaturation values of MTase in the presence and absence of Mg2+. This revealed changes in the structural conformational and stability of MTase at pH 8.0.

**Table 1.** Percentage change in secondary structure content and thermal denaturation values of


mid-point (Tm) by fitting the ellipticity. The results showed that secondary structural contents of MTase were transformed during the thermal denaturation process. The loss of protein function and structure were directly related to the decrease in the ellipticity at 222 nm with temperature. The obtained Tm values of native MTase were calculated at around ~58.8 °C, which changed significantly compared to the presence of Mg2+ (Figure 8B, Table 1). The thermal denaturation of MTase and waning of hydrophobic and other non-covalent interactions might be responsible. *2.5. Determination of Binding Affinity and Mechanism of Mg2+ Ion with MTase by Fluorescence Quenching Method*  We calculated the thermal unfolding of MTase by the two-state folding–unfolding model and the related Equations (2) and (3), which are used to resolve the temperature mid-point (Tm) by fitting the ellipticity. The results showed that secondary structural contents of MTase were transformed during the thermal denaturation process. The loss of protein function and structure were directly related to the decrease in the ellipticity at 222 nm with temperature. The obtained T<sup>m</sup> values of native MTase were calculated at around ~58.8 ◦C, which changed significantly compared to the presence of Mg2+ (Figure 8B, Table 1). The thermal denaturation of MTase and waning of hydrophobic and other noncovalent interactions might be responsible.

#### affinity of Mg2+ with MTase. For this, we titrated Mg2+ against MTase (5 μM) at 25 °C. Figure 9A shows that MTase possesses a sharp fluorescence emission peak around 340 *2.5. Determination of Binding Affinity and Mechanism of Mg2+ Ion with MTase by Fluorescence Quenching Method*

We also performed fluorescence quenching experiments to determine the binding

nm when excited at 280 nm. The quenching of MTase fluorescence occurs by increasing Mg2+ to its saturation level, at a millimolar concentration. It is possible that Mg2+ intercalates with MTase at a site close to tryptophan or other aromatic amino acid residues. This region is predominantly responsible for the change in the emission peak at around 340 nm after excitation at 280 nm [25]. Therefore, an ongoing reduction in the emission spectral intensity of MTase has been found, without remarkable variation in the wavelength We also performed fluorescence quenching experiments to determine the binding affinity of Mg2+ with MTase. For this, we titrated Mg2+ against MTase (5 µM) at 25 ◦C. Figure 9A shows that MTase possesses a sharp fluorescence emission peak around 340 nm when excited at 280 nm. The quenching of MTase fluorescence occurs by increasing Mg2+ to its saturation level, at a millimolar concentration. It is possible that Mg2+ intercalates

of maximal fluorescence emission (λmax) until the final quenching concentration [26].

with MTase at a site close to tryptophan or other aromatic amino acid residues. This region is predominantly responsible for the change in the emission peak at around 340 nm after excitation at 280 nm [25]. Therefore, an ongoing reduction in the emission spectral intensity of MTase has been found, without remarkable variation in the wavelength of maximal fluorescence emission (λmax) until the final quenching concentration [26]. *Molecules* **2022**, *27*, x FOR PEER REVIEW 10 of 18

**Figure 9.** (**A**) Fluorescence quenching measurement: fluorescence emission spectra of MTase (5 μM) in the presence of Mg2+. (**B**) Stern–Volmer plot for the MTase–Mg2+ interaction. (**C**) Binding parameter measurements: plot of log [(Fo/F) − 1] vs. log [Mg2+] for the determination of binding constants and binding stoichiometry for the MTase–Mg2+ interaction at room temperature and pH 8.0. **Figure 9.** (**A**) Fluorescence quenching measurement: fluorescence emission spectra of MTase (5 µM) in the presence of Mg2+. (**B**) Stern–Volmer plot for the MTase–Mg2+ interaction. (**C**) Binding parameter measurements: plot of log [(Fo/F) <sup>−</sup> 1] vs. log [Mg2+] for the determination of binding constants and binding stoichiometry for the MTase–Mg2+ interaction at room temperature and pH 8.0.

To determine the binding affinity of MTase, we performed the titration of Mg2+ against MTase at 25 °C. However, the *k*q value for the MTase–Mg2+ system was ten times higher than the highest scatter collision quenching constant of innumerable quenchers with polymers (2 × 1010 M−1s−1) [27]. This reflects that quenching is not commenced by dynamic diffusion, but arises by creating powerful complex formation between MTase and Mg2+. To determine the binding affinity of MTase, we performed the titration of Mg2+ against MTase at 25 ◦C. However, the *k*<sup>q</sup> value for the MTase–Mg2+ system was ten times higher than the highest scatter collision quenching constant of innumerable quenchers with polymers (2 <sup>×</sup> <sup>10</sup><sup>10</sup> <sup>M</sup>−<sup>1</sup> s −1 ) [27]. This reflects that quenching is not commenced by dynamic diffusion, but arises by creating powerful complex formation between MTase and Mg2+ .

The intrinsic fluorescence intensity (FI) of aromatic amino acids decreases continuously by increasing the metal ion concentration. For example, the emission spectra become saturated at 30 mM Mg2+, as shown in Figure 9A. The decrease in FI upon adding ions was analysed using the Stern–Volmer equation. It is a fact that the slopes in Figure 9B indicate that the binding of the ligand to the protein is responsible for quenching [28]. Based on the Stern–Volmer plot, the *K*q value of Mg2+ is 5.81 × 109, reflecting lower scatter collision quenching value initiated by the dynamic diffusion of molecules. The intrinsic fluorescence intensity (FI) of aromatic amino acids decreases continuously by increasing the metal ion concentration. For example, the emission spectra become saturated at 30 mM Mg2+, as shown in Figure 9A. The decrease in FI upon adding ions was analysed using the Stern–Volmer equation. It is a fact that the slopes in Figure 9B indicate that the binding of the ligand to the protein is responsible for quenching [28]. Based on the Stern–Volmer plot, the *<sup>K</sup>*<sup>q</sup> value of Mg2+ is 5.81 <sup>×</sup> <sup>10</sup><sup>9</sup> , reflecting lower scatter collision quenching value initiated by the dynamic diffusion of molecules.

The binding constant and related binding stoichiometry of Mg2+ were calculated as per log [(Fo/F) − 1] plotted against log [Mg2+], as presented in Figure 9C. The slope of these plots reveals that binding stoichiometry (*n*) and corresponding intercept value give the information about binding constant (*K*b), which was calculated from Equation (5), with computed values are reflected in Table 2. The binding constant and related binding stoichiometry of Mg2+ were calculated as per log [(Fo/F) <sup>−</sup> 1] plotted against log [Mg2+], as presented in Figure 9C. The slope of these plots reveals that binding stoichiometry (*n*) and corresponding intercept value give the information about binding constant (*K*b), which was calculated from Equation (5), with computed values are reflected in Table 2.

**Table 2.** Binding parameters for the MTase and metal (Mg2+) ion complex at standard temperature (25 °C). **Table 2.** Binding parameters for the MTase and metal (Mg2+) ion complex at standard temperature (25 ◦C).


to other pH and more feasible for the study of interaction with other inhibitors in the presence of Mg2+. These consequences occur because of the pH-induced alteration in the vicinity of the metal-binding site of the MTase that is responsible for the change in the mode and mechanism of protein, and finally, the interaction of MTase with Mg2+ [29]. *2.6. Isothermal Titration Calorimetry (ITC) Analysis*  Therefore, our CD and fluorescence data imply that under slightly basic conditions (at pH 8.0), the structural and conformational alteration in MTase was minimal compared to other pH and more feasible for the study of interaction with other inhibitors in the presence of Mg2+. These consequences occur because of the pH-induced alteration in the vicinity of the metal-binding site of the MTase that is responsible for the change in the mode and mechanism of protein, and finally, the interaction of MTase with Mg2+ [29].

(at pH 8.0), the structural and conformational alteration in MTase was minimal compared

#### The injected heat signals for Mg2+ binding with MTase and the integrated heat of the reac-*2.6. Isothermal Titration Calorimetry (ITC) Analysis*

tion for each injection are displayed in Figure 10. The calculated output results of the ITC data are shown in Table 3, which demonstrate that there are two binding sites available for Mg2+ in MTase. The first binding (6.7 × 104 M−1) site is much stronger, which is driven The thermodynamic parameters of Mg2+ upon binding to MTase were studied by ITC. The injected heat signals for Mg2+ binding with MTase and the integrated heat of the reaction

The thermodynamic parameters of Mg2+ upon binding to MTase were studied by ITC.

MTase– Mg2+

for each injection are displayed in Figure 10. The calculated output results of the ITC data are shown in Table 3, which demonstrate that there are two binding sites available for Mg2+ in MTase. The first binding (6.7 <sup>×</sup> <sup>10</sup><sup>4</sup> <sup>M</sup>−<sup>1</sup> ) site is much stronger, which is driven by a small negative enthalpy (−0.08 ± 0.06 kcal/mol) and a large positive entropy (6.49 ± 0.10 kcal/mol). The favourable entropy contributes to the solvation entropy because of the loss of water from the binding interface, indicating the presence of electrostatic interaction during the complex formation. Meanwhile, the secondary binding site is weaker (3.0 <sup>×</sup> <sup>10</sup><sup>2</sup> <sup>M</sup>−<sup>1</sup> ) and endothermic. Based on the thermodynamic analysis, it is suggested that the protein–Mg2+ complexation is mainly driven by electrostatic interaction. by a small negative enthalpy (−0.08 ± 0.06 kcal/mol) and a large positive entropy (6.49 ± 0.10 kcal/mol). The favourable entropy contributes to the solvation entropy because of the loss of water from the binding interface, indicating the presence of electrostatic interaction during the complex formation. Meanwhile, the secondary binding site is weaker (3.0 × 102 M−1) and endothermic. Based on the thermodynamic analysis, it is suggested that the protein–Mg2+ complexation is mainly driven by electrostatic interaction.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 11 of 18

**Figure 10. ITC thermogram:** ITC thermogram showing the binding of protein with Mg2+. The experiment was performed at 25 °C; top panel, detected heat signals; bottom panel, integrated heat of reaction for each titration. **Figure 10. ITC thermogram:** ITC thermogram showing the binding of protein with Mg2+. The experiment was performed at 25 ◦C; top panel, detected heat signals; bottom panel, integrated heat of reaction for each titration.


**Table 3.** Thermodynamic parameters obtained from ITC thermogram.

± 1.15 × 104 <sup>−</sup>0.08 ± 0.06 6.49 ± 0.10 <sup>−</sup>6.57 ± 0.16 300 ± 55 4.00 ± 0.36 7.39 ± 0.44 <sup>−</sup>3.39 ± 0.09 *2.7. Binding Study of Magnesium with MTase*

approximately 15 μM (Figure 11).

*2.7. Binding Study of Magnesium with MTase*  Further, the binding affinity of magnesium and MTase was determined using microscale thermophoresis (MST), a tool to study the interaction of the biomolecules. During the study, the MTase concentration was set at 50 nM, and the concentration of Mg2+ was varied from 5 mM to 300 nM in 16 different dilutions. The graph was plotted using the concentration of Mg2+ on the *X*-axis in log [M], while the *Y*-axis displayed the normalised fluorescence. The sample fluorescence is recorded during an MST experiment, starting with 3 s at ambient temperature to monitor steady-state fluorescence, followed by IR laser activation for a defined MST-on time. The MST analysis was performed using MO Control and MO Affinity Analysis Software (Monolith NT.115, NanoTemper Technologies, Mün-Further, the binding affinity of magnesium and MTase was determined using microscale thermophoresis (MST), a tool to study the interaction of the biomolecules. During the study, the MTase concentration was set at 50 nM, and the concentration of Mg2+ was varied from 5 mM to 300 nM in 16 different dilutions. The graph was plotted using the concentration of Mg2+ on the *X*-axis in log [M], while the *Y*-axis displayed the normalised fluorescence. The sample fluorescence is recorded during an MST experiment, starting with 3 s at ambient temperature to monitor steady-state fluorescence, followed by IR laser activation for a defined MST-on time. The MST analysis was performed using MO Control and MO Affinity Analysis Software (Monolith NT.115, NanoTemper Technologies, München, Germany). The calculated dissociation constant (KD) between MTase and Mg2+ is approximately 15 µM (Figure 11).

chen, Germany). The calculated dissociation constant (KD) between MTase and Mg2+ is

**Figure 11. Microscale thermophoresis:** Dose–response curve for the binding interaction between RED-Tris-NTA-labelled MTase and Mg2+. The concentration of RED-Tris-NTA-labelled MTase is constant, while the concentration of Mg2+ varies between 5 mM and 300 nM. The KD value for MgCl2 with MTase is 15 μM. The *Y*-axis on the graph represents the fluorescence change, and the *X*-axis on the graph represents the concentration of Mg2+. The graph describes the values of three different **Figure 11. Microscale thermophoresis:** Dose–response curve for the binding interaction between RED-Tris-NTA-labelled MTase and Mg2+. The concentration of RED-Tris-NTA-labelled MTase is constant, while the concentration of Mg2+ varies between 5 mM and 300 nM. The K<sup>D</sup> value for MgCl<sup>2</sup> with MTase is 15 µM. The *Y*-axis on the graph represents the fluorescence change, and the *X*-axis on the graph represents the concentration of Mg2+. The graph describes the values of three different experiments.

### experiments. **3. Discussion**

**3. Discussion**  The plus-stranded viruses have a 5′-capped genome catalysed by MTase [30,31] and found to be essential for their infectivity and replication [6,7]. In line with this, the 5′ noncoding region (NCR) of HEV RNA has been demonstrated to have an m7G-cap that is indispensable for its life cycle [6,32]. Similar results have been reported in alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus nonstructural protein [8]. Despite being an important enzyme that may act as a drug target, not many structural or functional studies have been conducted on MTase. Previously, the molecular weight of the active enzyme has been demonstrated to be 110 kDa, encoding amino acids 1 to 979 of the HEV genome [8]. In another study, using a computational approach, the MTase region has been predicted to be from 56 to 240 amino acids [9]**.** Emerson et al.'s computational predictions revealed that a region of 33–353 amino acids on the HEV pSK-HEV2 genome could exhibit MTase activity [6]. Therefore, we expressed this region to translate a protein of 37 kDa in size, as confirmed by western blotting and MALDI-TOF (Supplementary data). A recent study also demonstrated that a ~37 kDa MTase enzyme was processed from the HEV-ORF1 polyprotein when Huh7 cells were transduced with BacMam-HEV [33]. In our previous study, the digestion of ORF1 polyprotein, using cysteine protease, yielded a ~37 kDa protein, detected by MTase epitope-specific antibodies [34]. The enzyme was thus expressed and found to be active, as determined using the luminescence-based assay. The activity was altered by various The plus-stranded viruses have a 50 -capped genome catalysed by MTase [30,31] and found to be essential for their infectivity and replication [6,7]. In line with this, the 5 <sup>0</sup> non-coding region (NCR) of HEV RNA has been demonstrated to have an m7G-cap that is indispensable for its life cycle [6,32]. Similar results have been reported in alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus nonstructural protein [8]. Despite being an important enzyme that may act as a drug target, not many structural or functional studies have been conducted on MTase. Previously, the molecular weight of the active enzyme has been demonstrated to be 110 kDa, encoding amino acids 1 to 979 of the HEV genome [8]. In another study, using a computational approach, the MTase region has been predicted to be from 56 to 240 amino acids [9]. Emerson et al.'s computational predictions revealed that a region of 33–353 amino acids on the HEV pSK-HEV2 genome could exhibit MTase activity [6]. Therefore, we expressed this region to translate a protein of 37 kDa in size, as confirmed by western blotting and MALDI-TOF (Supplementary data). A recent study also demonstrated that a ~37 kDa MTase enzyme was processed from the HEV-ORF1 polyprotein when Huh7 cells were transduced with BacMam-HEV [33]. In our previous study, the digestion of ORF1 polyprotein, using cysteine protease, yielded a ~37 kDa protein, detected by MTase epitope-specific antibodies [34]. The enzyme was thus expressed and found to be active, as determined using the luminescencebased assay. The activity was altered by various cap analogues, as seen in an earlier study by Magden et al. [8]. The enzyme activity was also confirmed using enzyme kinetics and binding studies.

cap analogues, as seen in an earlier study by Magden et al. [8]. The enzyme activity was also confirmed using enzyme kinetics and binding studies. Another objective of this study is the metal dependency of MTase activity and several viral MTases, as other enzymes use divalent cations for their activity [17–20], which prompted us to study the effect of Ca2+ and Mg2+ on MTase activity. While there was no considerable effect on the enzyme activity was observed in the presence of Ca2+ (data not shown), significant MTase activity was observed in the case of Mg2+. The results confirmed that activity was decreased when Mg2+ was depleted by the chelating agent, EDTA. In Another objective of this study is the metal dependency of MTase activity and several viral MTases, as other enzymes use divalent cations for their activity [17–20], which prompted us to study the effect of Ca2+ and Mg2+ on MTase activity. While there was no considerable effect on the enzyme activity was observed in the presence of Ca2+ (data not shown), significant MTase activity was observed in the case of Mg2+. The results confirmed that activity was decreased when Mg2+ was depleted by the chelating agent, EDTA. In eukaryotes, mRNA capping also requires Mg2+ for the catalysis of lysine–GMP intermediate formation [35]. The metal ions are known to stabilise the random coil regions of enzymes by forming metal-binding pockets and protecting the unstructured part from the protease

activity; they act as electron donors at the catalytic centre to expand the biochemical palette and regulate a wide range of functions.

In this work, we performed biophysical studies to explore the importance of Mg2+ , which affects the stability of MTase at different ranges of pH and temperature. The far-UV-CD spectra (190–250 nm) clearly showed that MTase contains both α-helices (27%), and β-sheets (23%), but different pH environments induced changes in the secondary structure components. The similar secondary structural components were seen in most SAM-dependent MTases with a Rossmann-like fold. Further, the CD experiments at different Mg2+ concentrations revealed that the secondary structure was proportional to Mg2+ concentration, which is evident in metal-binding proteins. Thermal denaturation experiments in the presence of Mg2+ were also performed to understand the effect of metal ions on MTase folding by increasing the temperature. Analysis of the plot revealed the reduction in the percentage of the secondary structure with increased temperature, indicating the absence of any intermediate unfolding states during the thermal unfolding pathway of MTase. The obtained T<sup>m</sup> values of MTase were calculated using native conditions that significantly increased in the presence of Mg2+. The waning of hydrophobic and polar interactions might be responsible for the denaturation of MTase with Mg2+; without it, MTase is very unstable and is precipitated. The binding affinity of Mg2+ determined using the fluorescence quenching experiment identified its strong association with MTase. The decrease in the emission intensity at 340 nm is reflected when increasing concentration of the Mg2+ , which are accountable for the quenching of fluorescence intensity from the aromatic amino acids closely associated with Mg2+ (Figure 9). The ITC experiments revealed that MTase has two Mg2+ binding sites. The first one is stronger, and entropy plays a significant role in the binding (Table 2); the second is weaker and endothermic. Therefore, ITC experiments suggested that the MTase–Mg2+ complexation is mainly driven by electrostatic interaction. MST analysis was also performed to determine the Mg2+ binding with MTase, and these results suggested that the Mg2+ ion has an affinity towards MTase.
