Figure 2.

Enzymatic activity of the B. subtilis CdaA_CDenzyme is inhibited by GlmM or GlmM_F369.A, metal dependency of the B. subtilis CdaA_CD enzyme. The metal dependency of the B. subtilis CdaA_CD was assessed by performing enzyme reactions using 5 μM of purified CdaA_CD in buffer containing 10 mM Mn⁺², Mg⁺², or Co⁺² ions. After 4 h of incubation, the reactions were stopped, separated by TLC, and the percentage conversion of radiolabeled ATP to c-di-AMP determined. The average values and standard deviations (SDs) from three experiments were plotted. B, CdaA_CD activity time-course experiment. Enzyme reactions were set up with the B. subtilis CdaA_CD enzyme in buffer containing 10 mM Mn⁺², aliquots removed, and reactions stopped at the indicated time points and separated by TLC. The percentage conversion of radiolabeled ATP to c-di-AMP was determined and the average values and SDs from three independent experiments plotted. C, enzyme activity of the B. subtilis CdaA_CD enzyme in the presence of B. subtilis GlmM. The enzyme activity of CdaA_CD was measured in the absence or presence of GlmM or GlmM_F369 at a molar ratio of 2:1 GlmM or GlmM_F369 to CdaA_CD. After 4 h of incubation, the reactions were stopped, separated by TLC, and the percentage conversion of radiolabeled ATP to c-di-AMP was determined. The average values and SDs from six independent experiments were plotted. One-way ANOVA tests followed by Dunnett’s multiple comparison were performed to identify statistically significant differences in cyclase activity in the absence or presence of GlmM or GlmM_F369. ∗∗∗ indicates p < 0.0001.