Extended Data Figure 4 |. Detailed structural analysis of Em-CdnE.

a, Sequence alignment of CdnE homologs in Fig. 2c, annotated with Rm-CdnE secondary structure features. Mg²⁺ coordinating active site residues are highlighted in red, and the analogous residues to Rm-CdnE N166 are highlighted in orange. WP_050915017, Yersinia enterocolitica; WP_096075289, Pseudomonas aeruginosa; WP_104644370, Xanthomonas arboricola; WP_010848498, Xenorhabdus nematophila; WP_015040391, Bordetella parapertussis; WP_006482377, Burkholderia cepacia complex; WP_014072508, Rhodothermus marinus (Rm-CndE); WP_042646516, Legionella pneumophila; WP_062886322, Mycobacterium avium; WP_016200549, Elizabethkingia meningoseptica (Em-CdnE); WP_031901603, Staphylococcus aureus; WP_050492554, Enterococcus faecalis; WP_062695386, Bacteroides thetaiotaomicron.

b, Biochemical deconvolution of Em-CdnE, which has a natural serine substitution at the N166 analogous site. Recombinant protein was incubated with NTPs as indicated and reactions were visualized as in Fig. 1b. Data are representative of 3 independent experiments.

c, Reactions of Em-CdnE incubated with α−³²P radiolabeled NTPs and nonhydrolyzable nucleotide analogs as indicated, and visualized as in Fig. 1b. Data are representative of 3 independent experiments.

d, Anion exchange chromatography of an Em-CdnE 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.

e, Anion exchange chromatography (IEX) fractions from d were separated by silica TLC, visualized by UV shadowing, and compared to a radiolabeled reaction to confirm the appropriate peak. Fractions were pooled and concentrated prior to MS analysis. MS confirmed synthesis of products with masses corresponding to c-di-AMP, cGAMP, and c-di-GMP.

f, Crystal structure of Em-CdnE in complex with GTP and nonhydrolyzable ATP capturing the “1^st state” structure prior to NTP hydrolysis. Mg²⁺ ions are omitted for clarity.

g, Zoom-in cut-away of the active site of f, confirming position of a serine at the analogous site to Rm-CdnE N166. Mg²⁺ ions are shown in green. Nucleotide and metal 2F_o−F_c electron density is contoured at 1 σ.

h, Zoom-in cut-away of the active site of Em-CdnE–pppApA structure, capturing the “2^nd state” after the first reaction has occurred to form a linear intermediate, but prior to CDN formation. Mg²⁺ ions are shown in green. Nucleotide and metal 2F_o−F_c electron density is contoured at 1 σ.

i, Biochemical deconvolution of mutant Em-CdnE reverted to the ancestral asparagine at the N166 analogous site. This mutant loses preference for producing cyclic dipurine molecules and instead produces more pyrimidine-containing CDN products. Reactions were visualized as in Fig. 1b. Data are representative of 2 independent experiments.