Figure 4. TDE1909 requires Mn⁺² or Co⁺² for DAC activity.

(A) Recombinant TDE1909 was incubated with 150 µM ATP for 4 hr in buffer containing 10 mM of MnCl₂, CoCl₂, or MgCl₂ and reactions products were analyzed by RP-HPLC. Purified ATP and c-di-AMP were fractionated as controls to determine elution profiles for each nucleotide. (B) Residues G171 and S221 were substituted with alanine by Q5 site-directed mutagenesis (NEB) and recombinant proteins with amino acids substitutions were purified and incubated with 150 µM ATP for 4 hr. The native TDE1909 protein was also run as a control along with purified ATP and c-di-AMP as controls.

Data shown is representative of duplicate experiments.