**4. Conclusions**

Briefly, we expressed MTase and determined its activity in its shortest functional form, 33–353 amino acids of HEV polyprotein, which was previously predicted by computational studies. The active MTase was determined to be 37 kDa in size by MALDI-TOF. The activity of the enzyme was confirmed using enzymatic assay, with SAM as a methyl donor and GTP as a methyl acceptor. GDP showed the highest activity and GTP showed the second highest as a methyl acceptor in the MTase activity assay. The activity of the enzyme was found to be increased in the presence of Mg2+, which is a feature of many RNA capping enzymes. Similarly, the activity of MTase was decreased when it was chelated with EDTA. The circular dichroism, fluorescence quenching, and thermal denaturation studies provided the MTase with structural stability in the presence of Mg2+. The binding affinity of Mg2+ with MTase was determined by ITC and MST experiments. Overall, our study has shown the indispensable role of Mg2+ in MTase activity and stability. Further, this work established the optimal experimental conditions that would be helpful for the screening of inhibitor libraries against HEV MTase to identify potential inhibitors.
