# The World of Cyclic Dinucleotides in Bacterial Behavior
### DAC Domain Structure and Catalysis  
DAC structure and structural similarities with other protein domains. c-di-AMP is synthesized by DAC enzymes that convert two molecules of ATP into one c-di-AMP and two pyrophosphate molecules. In the case of DisA, the Rv3586 protein from _Mycobacterium tuberculosis_, the synthesis of c-di-AMP is made using ATP or ADP [117]. Production of c-di-AMP has been described as essential for the growth of some Gram-positive bacteria due to it being involved in crucial cellular activities, such as cell wall metabolism, maintenance of DNA integrity, ion transport, cell division, and cell size control [118; 120; 121; 122]. _Bacillus subtilis_ encodes three DAC enzymes, DisA, CdaA, and CdaS. Two of them, DisA and CdaA, are constitutively expressed during vegetative growth while CdaS is required for efficient germination of spores. Other Gram-positive bacteria encode only one DAC protein that is essential for their growth, as observed in _Listeria monocytogenes_, _Streptococcus pneumoniae_, and _Staphylococcus aureus_, thus making this enzyme a likely target for constructing new inhibitors that may serve as antibiotics for pathogenic Gram-positive bacteria. At the moment, three structures of proteins containing DAC domains have been solved: DisA from _Thermotoga maritima_ (PDBID 3C1Y) [3], DisA (named in the UniProt database as DacB) from _Bacillus cereus_ (PDBID 2 FB5) [121]; and DacA (CdaA-APO Y187A Mutant) from _L. monocytogenes_ (PDBID 6 HVN) [122]. As described before, no structural similarities with other domain were detected so far. The overall DAC domain structure exhibits a globular \(\alpha\)/\(\beta\) fold with a slightly twisted central \(\beta\)-sheet, made up of seven mixed-parallel and antiparallel \(\beta\)-strands (\(\beta\)1-\(\beta\)7) surrounded by five \(\alpha\)-helices (\(\alpha\)1-\(\alpha\)5), in which the N-terminal helix (\(\alpha\)1) can be split in two parts (\(\alpha\)1' and \(\alpha\)1). Like GGDEF domains, two DAC domains must be correctly oriented to allow the conversion of two ATP molecules into one c-di-AMP molecule and two pyrophosphates. Therefore, DAC domains are also Bi Ter enzymes (two substrates, three products). The regulation of the catalytic activity of DAC domains may be regulated by input domains (Figure 2C), and in the case of DisA from _T. maritima_ (PDB code 3C23, 3C1Z, and 3C1Y) [3], the protein forms a homo-octamer and the DAC domains are oriented in a such way that two DAC domains are oriented face to face to allow the catalysis. Therefore, in each DisA homo-octamer, there are four potential catalytic sites. Linear DNA or DNA ends do not affect the protein activity but branched nucleic acids (such as in Holliday junctions) strongly suppress the DAC activity of DisA by binding to its C-terminal domain [3]. Active site of DAC domains--In the case of the DAC domain of CdaA from _Listeria monocytogenes_ (PDBID 4 RV7), the ATP ligand is located in a well-defined cavity made up by the N-terminus of \(\alpha\)4, loop \(\beta\)5-\(\beta\)6, loop \(\beta\)4-\(\alpha\)4, and loop \(\alpha\)3-\(\beta\)3 (Figure 6C), in which many conserved residues of DAC domains are located: the GALI motif, GxRHRRxA motif, an absolutely conserved serine, DGAhh motif (h is a hydrophobic residue), and (V/I)SEE motif (Figure 6A). The active site of CdaA DAC domain is thought to be built by 10 residues: the side chains of Leu31, Asp71, Thr102, Arg103, His104, Ser122, and Glu124 and the main chain of Leu88, Gly101, and Glu123 (Figure 6C). Leu31 belongs to the GALI motif, the Asp71 belongs to the DGAhh motif, and Thr102, Arg103, and His104 belong to GxRHRRxA motif. The main chain of Leu88 and the side chain of L31 interact with the adenine base, Asp71 binds the ribose, and Thr102, Arg103, His104, Ser122, Gly101, and Glu123 bind the phosphate groups of the ATP molecule. The Glu124 coordinates one magnesium cation that binds the \(\alpha\) and \(\beta\) phosphate groups of the ATP molecule (Figure 6C) [49]. CdaA from _L.