Analysis of kinase activity of mutant proteins
Kinase activity of the mutant proteins was assessed to check the most essential domain for the activity of SK using ADP-Glo kinase assay. Assay was performed using 2µg/ml of shikimic acid and 600 ng of purified protein. Comparative analysis of the kinase activity of mutant proteins was done with respect to purified recombinant SK. The result shows >90% decrease in activity of protein where CD was mutated (CD15:SK) while, around 50% decrease was observed in the protein where LD was manipulated (LD117:SK). Moreover, no effect was observed in kinase activity of the protein where SBD was mutated (SBD34:SK). Overall findings suggest that the activity of the enzyme decreases significantly upon mutation of CD, depicting its vital role in enzyme activity whereas SBD34:SK does not have any effect on enzymatic activity (Figure 2D).