Extended Data Figure 9 |. Detailed biochemical analysis of Ec-CdnD02.

a-, Titration of reaction buffer pH in steps of 0.2 pH units. Recombinant Ec-CdnD02 was incubated with α−³²P radiolabeled NTPs at varying pH and the reactions were analyzed and visualized by PEI-cellulose or silica TLC as in Fig. 1b and Fig. 4c. Silica TLC identified two products, denoted the major (blue triangle) and minor (red triangle) product. Quantification of TLC spots is shown below. Data are representative of 2 independent experiments.

b-, Biochemical deconvolution of Ec-CdnD02, recombinant protein was incubated with NTPs as indicated and analyzed by TLC as in a. Data are representative of 3 independent experiments.

c-, Nuclease digestion of the Ec-CdnD02 product. Conventional nuclease digestion includes addition of a phosphatase, in this experiment reactions were first treated with Antarctic phosphatase to remove remaining NTPs then heat inactivated. Next, reactions were either untreated, treated with nuclease P1 (specific for 3′–5′ phosphodiester bonds) only, or treated with nuclease P1 and phosphatase to remove exposed phosphate groups. 3′3′ cGAMP (DncV) and Ec-CdnD02 product are digested into AMP and GMP constituents, which are phosphatase sensitive. cAMP (CyaA) is insensitive to P1 digestion and cyclic monophosphates are phosphatase resistant. These data demonstrate that the Ec-CdnD02 product does not contain a cyclic monophosphate. Data are representative of 3 independent experiments.

d, Incubation of Ec-CdnD02 with nonhydrolyzable nucleotides, as described in Extended Data Fig. 2. Nonhydrolyzable ATP completely blocks the reaction, indicating the first step requires attack of the α-P from ATP. However, when nonhydrolyzable GTP is present the possible intermediates [pp(c)pGpA, pp(c)pGpApA, or pppApA] cannot be distinguished in this assay and it is unclear how the reaction proceeds. Silica TLC is not suited for analyzing nonhydrolyzable nucleotides because they do not migrate beyond the origin. Data are representative of 3 independent experiments.

e, Anion exchange chromatography of a Ec-CdnD02 reaction with ATP and GTP, eluted with a gradient of Buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated prior to pooling for further analysis.

f and g, 3′3′3′ tricyclic adenosine monophosphate–adenosine monophosphate–guanosine monophosphate (cAAG) NMR spectra and associated zoomed in dataset. ³¹P{¹H} NMR (162 MHz): δ_P −0.65 (s, 1P), −0.70 (s, 1P), −0.75 (s, 1P).

h-j, 3′3′3′ tricyclic adenosine monophosphate–adenosine monophosphate–guanosine monophosphate (cAAG) proton NMR spectra and associated zoomed in datasets. ¹H NMR (400 MHz): δ_Η 8.43 (s, 1H), 8.39 (s, 1H), 8.19 (s, 1H), 8.12 (s, 1H), 8.01 (s, 1H), 6.15 (d, J = 7.0 Hz, 1H), 6.12 (d, J = 7.0 Hz, 1H), 5.92 (d, J = 7.5 Hz, 1H), 5.00–4.78 (m, 6H), 4.69–4.58 (m, 3H), 4.3–4.2 (m, 6H).