Figure 7. TDE0027 is a phosphodiesterase that hydrolyzes c-di-AMP into pApA.

(A) Recombinant TDE0027 was incubated with either bis-pNPP or pNPP in buffer containing MnCl₂ or MgCl₂ for 30 min. Hydrolysis of both substrates was monitored at 405 nm. Data are the average from 3 replicates with standard deviation analyzed by one-way ANOVA and Dunnett’s post-hoc test. (B) Hydrolysis of bis-pNPP after 30 min was measured at 405 nm with increasing amounts of TDE0027 enzyme (n=3). (C) The hydrolysis of bis-pNPP was monitored at 405 nm every 5 min with various amounts the TDE0027 enzyme (n=3). (D) The products generated after incubating TDE0027 with c-di-AMP and pApA for 4 hr were identified by RP-HPLC. As a control, TDE0027 was incubated with c-di-AMP in a buffer containing 10 mM EDTA. Purified AMP, c-di-AMP and pApA were also fractionated to determine retention times in the column. Data are representative of duplicate experiments.