## 2 Bacterial cON PDEs as Potential Drug Targets
### Eal PDEs  
EAL PDEs are widespread in bacteria and are often associated with sensory domains such as GGDEF (modified), PAS, REC, or GAF, which allow them to respond to environmental signals (Figure 4A) [39; 40; 41; 42; 43]. Interestingly, some EAL domain proteins, such as FimX from _Pseudomonas aeruginosa_, are catalytically inactive and instead function as high-affinity c-di-GMP receptors [44]. The regulation of EAL domain activity is further influenced by environmental factors such as pH, metal ion concentration, and even light in the case of photoreceptor proteins like BlrP1 [45]. Figure 3: Structures of notable cONs which are discussed in detail in this review. The structural characterization of EAL domain proteins has provided significant insights into their function. Crystal structures of proteins such as BlrP1 from _Klebsiella pneumoniae_ and TBD1265 from _Thiobacillus denitrificans_ have revealed that EAL domains adopt a variation in the classic (\(\alpha\)\(\beta\))8 TIM barrel fold [45; 49]. The active site is located at the C-terminal end of the barrel and is usually occupied by two divalent metal ions when bound to c-di-GMP. [PERSON] and co-workers first proposed the catalytic mechanism of EAL PDEs, using RocR from _P. aeruginosa_ and tdEAL from _T. denitrificans_[46]. They described a Mg\({}^{2+}\)-assisted, general base-catalyzed hydrolysis of c-di-GMP, where a conserved Glu in the EAL motif acts as a base catalyst, deprotonating a Mg\({}^{2+}\)-coordinated water molecule to generate a nucleophilic hydroxide ion for cleavage (Figure 4B). Later, the crystal structure of BLUF-EAL PDE Blrp1 from _Klebsiella pneumoniae_ in complex with its substrate led to the proposal of a two-metal ion mechanism. In this model, two metal ions are coordinated by essential carboxylate residues, including Glu 359 from the EAL motif, a bridging water molecule, and the phosphate oxygen of c-di-GMP (Figure 4C,D). Subsequently, a general two-metal-ion mechanism was proposed for EAL domain enzymes, similar to other nucleotide phospho-diseterases, where one metal ion activates a water molecule for nucleophilic attack, while the other stabilizes the transition state and facilitates leaving group formation [47; 48]. Figure 4: **(A)** A typical EAL PDE catalytic domain with a regulatory domain [39; 40; 41; 42; 43]. **(B)** Proposed Mg\({}^{2+}\)-assisted, general base-catalyzed hydrolysis of c-di-GMP [46]. **(C)** The crystal structure of BlrP1 (PDB: 3 GG0) reveals Mn\({}^{2+}\) ions (shown in purple) coordinated by multiple carboxylate residues. Among these, Glu359, positioned on strand \(\beta\)7 EAL, plays a key role, along with Asp302 and Asp303, which are located on a loop near the dimer interface. Additionally, the structure highlights a potential hydrolytic solvent molecule, HOH409, situated in a possible near-attack conformation, as well as HOH408, a water molecule that may function as a proton donor for the leaving group [45]. **(D)** Proposed two-metal mediated catalytic mechanism of c-di-GMP hydrolysis [47; 48]. Many EAL proteins function as dimers, and dimerization appears to be a crucial requirement for enzymatic activity [50]. Using biophysical and functional analyses, [PERSON] and co-workers showed that the isolated PDE EAL domain from _E. coli_ (Yaha) is in a fast thermodynamic equilibrium between a monomer and dimer, and that the EAL domain is only active in its dimeric form [51]. The rate of dimerization is shown to be increased by about 100-fold upon substrate binding, and specific site-directed mutagenesis preventing dimerization results in reduced PDE activity without changing the substrate binding affinity. This finding was corroborated by [PERSON] et al., whose recent structural analyses of EAL domains from _Pseudomonas aeruginosa_ PA1727 (MucReal) and PA3825 (PA3825 AL) revealed that EAL PDE activity is modulated by different dimerization interfaces [50].