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).