# The World of Cyclic Dinucleotides in Bacterial Behavior
### GGDEF Domain Structure and Catalysis
DGC: diguanylate cyclase; AC: adenylate cyclase, GC: guanylate cyclase, and FAP: 2-amino-5-formylamino-6-ribosylamino-4(3\(H\))-pyrimidinone 5'-phosphate. Residues are important to GGDEF catalysis. The GGDEF domains are diguanylate cyclases (DGCs) that convert two molecules of GTP into one molecule of c-di-GMP. The GGDEF active site is thought to be assembled only when two GGDEF domains come together in such a manner that permits the nucleophilic attack of the 3' OH groups on the \(\alpha\)-phosphate groups of each GTP, leading to the synthesis of one molecule of c-di-GMP and two pyrophosphate molecules [70; 81; 80]. Therefore, DGCs are Bi Ter enzymes (two substrates, three products) as described above [68; 69]. The catalytic activity of GGDEF domains is often regulated by input domains that precede the GGDEF domain, of which most are known or predicted to form dimers or heterodimers and to be sensor domains (Figure 2A). Isolated GGDEF domains have little or no detectable enzymatic activity [37; 82] and require the dimerization of the input domain to assemble a catalytically competent GGDEF domain. Two hypotheses of the GGDEF activity regulation were reported. One of them suggests that the input domain binds its ligand and enhances the homodimerization and consequently the correct orientation of GGDEF domains to perform the catalysis. The other hypothesis suggests that the protein is a homodimer already and, when the input domain binds its ligand, it causes a reorientation of the GGDEF domains to a catalytically competent GGDEF dimer, or vice versa [67]. The signal transduction from the input domain to the GGDEF domain is predicted to be relayed by a S-helix (signaling-helix) that connects the two domains and forms a two-helical parallel coiled coil (stalk) in the dimer form of the protein [67; 83]. Some proteins containing GGDEF domains possess a more complex activation mechanism and may involve formation of higher oligomers [67; 84; 85; 86]. The GG(D/E)EF motif (glycine, glycine, aspartic or glutamic acid, and phenylalanine residues) is located in the loop between \(\beta\)2 and \(\beta\)3 (Figure 3A), in which the glutamic acid residue binds to the \(\alpha\)-phosphate group of GTP molecule as well as coordinates one of the cations located in the binding site (Figure 3B). In the case of the PleD GGDEF domain, two magnesium cations are located in the binding site and are coordinated by E370 (from the GG(D/E)EF motif), D327, and the main chain of I328. The PleD residues D344 and N335 bind the guanosine base of the substrate, while the side chains of E370, K442, R446 and the main chains of F330, F331, and K332 bind the phosphate moieties of the GTP molecule (Figure 3B) [80]. The GG(D/E)EF consensus sequence and most of the residues important to catalysis are very well conserved within GGDEF family members (Figure 3A). This includes the D327, N335 and D344 residues, which have been reported to be essential to GGDEF domain activity [70; 87]. A subclass of GGDEF domain, called Hybrid promiscuous (Hypr) GGDEF enzymes, synthesizes predominantly cGAMP molecules, but also synthesizes c-di-AMP and c-di-GMP molecules [41]. The change in the substrate specificity seems to be related with the substitution of an aspartate (D344 of PleD located at the \(\alpha\)2) by a serine, exactly the residue that binds the guanine base of the GTP in PleD. Figure 2: Domain architectures of proteins containing GGDEF, SMODS (Second Messenger Oligonucleotide or Dinucleotide Syntheta  
Figure 1: Domain architectures of proteins containing GGDEF, SMODS (Second Messenger Oligonucleotide or Dinucleotide Syntheta  
Figure 1: Comparison of the results of the experiments on the GGDEF (**A**), SMODS (**B**) and DAC (**C**) domains. The analysis was done using a non-redundant dataset (<80% identity) of protein sequences built from sequences retrieved from the NCBI protein database [88]. The names of the domains are based on the Pfam database [89]. Figure 3: Conserved sequences within GGDEF members and their active and I-sites pockets.