## 2 Bacterial cON PDEs as Potential Drug Targets
### Hd-GYP PDEs  
HD-GYP phosphodiesterase(PDEs) are metal-dependent enzymes that belong to the HD phosphohydrolase superfamily but contain an additional GYP motif and are distinct from EAL-domain PDEs [54]. HD-GYP PDEs are assumed to be less widespread than EAL PDE and are often mutually exclusive with EAL-domain PDEs. Unlike EAL-domain PDEs, which hydrolyze c-di-GMP into linear pGpG, HD-GYPs can further degrade pGpG into GMP. The HD domain is characterized by a conserved catalytic dyad of histidine (H) and aspartate (D), which is necessary for coordinating divalent metal ions. Bioinformatics analysis shows that HD-GYP domains are prevalent in bacterial genomes, particularly in Proteobacteria and Firmicutes, but absent in archaea and eukaryotes [55]. The first identified HD-GYP PDE, RpfG from _Xanthomonas campetsris_, is well characterized for its role in c-di-GMP hydrolysis [56]. It is linked to a two-component regulatory system, where the sensor histidine kinase RpfC phosphorylates RpfG upon detecting quorum-sensing  
Figure 5: (**A**) A proposed dimerization mechanism regulating c-di-GMP hydrolysis catalyzed by EAL-domain PDE. A full-length EAL PDE, equipped with a regulatory (R) domain, undergoes a structural transition upon signal detection. The monomeric protein dimerizes through interactions between its R domains, which in turn facilitates the dimerization of the EAL domains, leading to the formation of an active PDE dimer. Once activated, this PDE dimer coordinates metal ions at the M1 and M2 sites via conserved aspartate residues, enabling substrate binding. A third metal ion occupies the M3 site, playing a crucial role in stabilizing the negatively charged transition state during the hydrolysis of c-di-GMP into pGpG [39; 40; 41; 42; 43]. (**B**) Crystal structure of CC3396\({}^{\text{EAL}}\) (PDB code: 3U2E) with manganese ions (purple) occupying M1, M2 and M3 sites [53]. (**C**) pGpG ligand structure showing the interactions with the coordinated metal ions [53]. signals, activating its PDE function. This activation reduces c-di-GMP levels, leading to the induction of virulence genes and inhibition of biofilm formation. Other bacterial species also exhibit HD-GYP-mediated regulation of biofilm formation and motility such as _Vibrio cholerae_, _Borrelia burgdorferi_, and _Pseudomonas aeruginosa_[57; 58; 59]. The HD-GYP domain adopts an all-\(\alpha\) fold with a catalytic metal-binding core. The GYP loop, which extends beyond the conserved HD motif, plays a role in substrate binding and possibly protein-protein interactions [59]. HD-GYP PDEs also form dimers, and dimerization may regulate enzymatic activity [60]. The PDE activity of HD-GYP proteins depends on the presence of metal ions, typically Mg\({}^{2+}\), Mn\({}^{2+}\), or Fe\({}^{2+}\), with some enzymes requiring a reduced di-iron center for function [61]. In _Pseudomonas marina_ PDE (PmGH), iron ions interact with metal-binding residues to stabilize the oxygen atoms within the central 12-membered ribose-phosphate ring of c-di-GMP. The Tyr285 residue, a key component of the GYP motif, plays a crucial role in strengthening binding and improving substrate specificity by forming a hydrogen bond with one of the nonbridging oxygen atoms in the ribose-phosphate ring (Figure 6). The presence of a third metal-binding residue, such as Glu185 in PmGH, determines whether the enzyme forms a trinuclear or binuclear metal center, influencing its ability to hydrolyze pGpG to GMP [62; 63]. The catalytic mechanism of HD-GYP PDEs is not yet fully understood. However, c-di-GMP hydrolysis appears to involve a metal-activated hydroxide ion that acts as a nucleophile and attacks the phosphorus atom of the ribose phosphate ring [59; 63]. Key residues in the active site, including those from the conserved KPG motif and the RXXK motif, help stabilize the substrate (Figure 6). Metal ions further facilitate the reaction by coordinating the oxygen atoms of the ribose phosphate ring and promoting bond cleavage [59; 63].