Extended Data Fig. 4: Detailed structural analysis of EmCdnE.

a, Sequence alignment of CdnE homologues in Fig. 2c, annotated with RmCdnE secondary structure features. Mg²⁺-coordinating active-site residues are highlighted in red and the analogous residues to RmCdnE Asn166 are highlighted in orange. Yersinia enterocolitica (WP_050915017); P. aeruginosa (WP_096075289); Xanthomonas arboricola (WP_104644370); Xenorhabdus nematophila (WP_010848498); Bordetella parapertussis (WP_015040391); Burkholderia cepacia complex (WP_006482377); R. marinus (RmCdnE, WP_014072508); L. pneumophila (WP_042646516); Mycobacterium avium (WP_062886322); E. meningoseptica (EmCdnE, WP_016200549); Staphylococcus aureus (WP_031901603); Enterococcus faecalis (WP_050492554); Bacteroides thetaiotaomicron (WP_062695386). b, Biochemical deconvolution of EmCdnE, which has a natural serine substitution at the Asn166 analogous site. Recombinant protein was incubated with NTPs as indicated and reactions were visualized as in Fig. 1b. Data are representative of three independent experiments. c, Reactions of EmCdnE incubated with α³²P-radiolabelled NTPs and non-hydrolysable nucleotide analogues as indicated and visualized as in Fig. 1b. Data are representative of three independent experiments. d, Anion exchange chromatography of an EmCdnE reaction with ATP and GTP, eluted with a gradient of buffer B (2 M ammonium acetate) by FPLC. Individual fractions were concentrated before pooling for further analysis. e, Anion exchange chromatography fractions from d were separated by silica TLC, visualized by ultraviolet-light shadowing and compared to a radiolabelled reaction to confirm the appropriate peak. Fractions were pooled and concentrated before mass spectrometry analysis. Mass spectrometry confirmed synthesis of products with masses corresponding to c-di-AMP, cGAMP and c-di-GMP. f, Crystal structure of EmCdnE in complex with GTP and non-hydrolysable ATP capturing the ‘first state’ structure before NTP hydrolysis. Mg²⁺ ions are omitted for clarity. g, Magnified cut-away of the active site of the complex shown in f, confirming the position of a serine at the analogous site to RmCdnE Asn166. Nucleotide and metal 2F_o − F_c electron densities are contoured at 1σ. h, Magnified cut-away of the active site of the EmCdnE–pppApA structure, capturing the ‘second state’ after the first reaction has occurred to form a linear intermediate, but before CDN formation. Nucleotide and metal 2F_o – F_c electron densities are contoured at 1σ. g, h, Mg²⁺ ions are shown in green. i, Biochemical deconvolution of mutant EmCdnE reverted to the ancestral asparagine at the Asn166 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 two independent experiments.