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.
Figure 2.
DAC activity is regulated by ATP.
(A) Optimum Mn2+ concentration. The optimum concentration of Mn2+ was determined by doing the assay (20 µl) in presence of 25 mM Tris-HCl (8.5), 25 mM NaCl, 300 µM ATP, 1 µM of MtbDisA, varying concentrations of MnCl2 and the reaction was incubated at 37°C for 15 min. The plot shows the amount (nmoles) of c-di-AMP formed during the reaction as a function of Mn2+ concentration (B) ATP dependence. The extent of formation of c-di-AMP in 20 µl reaction mixtures was determined by doing the DAC assay in presence of 25 mM Tris-HCl (8.5), 0.6 mM Mn2+, 25 mM NaCl, 1 µM of MtbDisA and different concentrations of ATP. The reaction mixtures were incubated at 37°C for 15 min. The plot shows the formation of c-di-AMP as a function of ATP concentration. The data plotted in (A) and (B) represent the mean of three independent experiments ± standard deviation. (C) ADP inhibition. The DAC assay was carried out in 10 µl reaction mixtures containing 25 mM Tris-HCl (pH 8.5), 0.6 mM MnCl2, increasing concentrations of ADP as indicated, 2 µM of MtbDisA and incubated for 15 min at 37°C. The reaction mixtures were processed and analyzed as before. The plot shows the formation of c-di-AMP as a function of ADP concentration. (D) D72AG73A has ATPase activity. The DAC assay was carried out in 10 µl reaction mixtures containing 25 mM Tris-HCl (pH 8.5), 0.6 mM MnCl2, indicated concentrations of ATP, 2 µM of D72AG73A and incubated for 30 min at 37°C. The reaction mixtures were processed and analyzed as before. The plot shows the formation of ADP as a function of ATP concentration.
Figure 3.
Detection of the intermediate ‘I’ of the DAC assay.
(A) LC - ESI -MS spectrum showing the peaks corresponding to proton and manganese ion adducts of the intermediate ‘I’ of molecular mass 836 Da. (B) Expansion of the region, m/z 442–452 of the spectrum shown in (A): m/z values and intensity distribution of isotope peaks indicating doubly charged manganese ion adduct of ‘I’.
Figure 4.
Reaction intermediate ‘I’ was determined to be pppApA.
(A) LC-ESI-MS/MS spectrum of [M+H]+ precursor ion m/z 837.05 (reaction intermediate ‘I’). (B) Figure depicting the interpretation of the fragmentation as observed in (A).
Figure 5.
Two-step synthesis of c-di-AMP.
The figure shows the formation of c-di-AMP from the two intermediates pppApA and ppApA. pppApA is first formed by conjugation between two molecules of ATP (step 1A). Two ADP molecules or one molecule of ADP and one ATP molecule can conjugate to give rise to ppApA (step 2A). pppApA and ppApA are then converted into c-di-AMP (steps 1B & 2B) through their respective Mn2+-adduct (transition state complex) as shown.
Figure 6.
c-di-AMP can exist as multimers.
The DAC reaction mixture was subjected to LC-ESI-MS and the mass spectra of the different multimeric forms have been shown here. (A) The LC-ESI mass spectrum showing signals corresponding to monomer, labeled ‘1′ and other multimeric forms, labeled ‘2′ and ‘3′ (B) Expansion of the region, m/z 648–672 of the spectrum in ‘A’ depicting isotope peaks corresponding to peak 1, which indicate presence of monomer and dimer (C) Expansion of the region, m/z 976–1000 of the spectrum in ‘A’, which shows isotope signals corresponding to peak 2 providing evidence for the presence of trimer of c-di-AMP (D) Expansion of the region, m/z 1306–1330 of the spectrum in ‘A’, indicating the presence of dimeric and tetrameric forms of c-di-AMP.
Figure 7.
Hydrolysis of c-di-AMP by MtbPDE.
The reaction mixtures of PDE assay were separated by reverse phase LC and the products were detected by measuring absorbance at 260–4 min have been shown here. The peaks have been labeled with the name of the eluted species. Number on the peak is the retention time of the species. (A) shows the hydrolysis product of c-di-AMP by MtbPDE. Inset shows the control reaction without the enzyme. (B) pApA was hydrolyzed by MtbPDE to AMP. Inset shows the control reaction without MtbPDE. (C) MtbPDE hydrolyzes ApA to AMP and adenosine (D) Hydrolysis of c-di-GMP by MtbPDE to 5′-GMP. Inset shows the control reaction without MtbPDE. (E) Mutant protein D130AH131A does not hydrolyze c-di-AMP.