Figure 1. MtbDisA synthesizes c-di-AMP through two intermediates.

DAC assay was done in 10 µl reaction mixtures containing 25 mM Tris-HCl (pH 8.5), 0.6 mM MnCl₂, 300 µM ATP and 2 µM of MtbDisA and incubated at 37°C. The reaction mixture was subjected to reverse phase LC and the products were detected with UV (λ 260 nm). The absorption chromatograms with peaks labeled with the corresponding species have been shown. (A) Peak for only ATP is detected in the control reaction (without enzyme). ATP is converted into c-di-AMP in the presence of MtbDisA as can be seen in (B). Two more peaks are detected in ‘B’ which are the intermediates of the reaction and are labeled ‘I’ and ‘II’. In (B) the DAC reaction was terminated after 5 min whereas in (C) and (D) the reaction was continued for 10 and 60 min respectively. Peak for ADP has been labeled.