## 9 DhhP-Type Phosphodiesterase  
DhhP-type phosphodiesterase are standalone DHH/DHHA1 domain proteins that degrade c-di-AMP in two steps: first converting it to pApA, then breaking it down into AMP (Figure 8A) [85; 87]. The crystal structure of Rv2837c (also called NmA and CdnP), a standalone DHH/DHHA1 domain protein from _Mycobacterium tuberculosis_ (Mtb), bound to 5\({}^{\prime}\)-pApA, a hydrolytic intermediate of c-di-AMP, provides valuable insights into the catalytic mechanism of DhhP-type phosphodiesterase (Figure 8B) [88]. The adenine bases of 5\({}^{\prime}\)-pApA are positioned perpendicularly, stabilized by \(\pi\)-\(\pi\) interactions with His312. Additionally, His312 forms a hydrogen bond with the phosphate group of the 3\({}^{\prime}\)-5\({}^{\prime}\) phosphodiester bond, which is coordinated by two Mn\({}^{2+}\) ions. This coordination facilitatesbond cleavage through a nucleophilic attack by an activated water molecule (Figure 8B). This mechanism is similar to the proposed two-metal catalytic mechanism for EAL PDEs (Figure 4D), and it is likely to be conserved across DHH-DHHA1 PDEs due to their shared binuclear metal center and substrate-binding residues. Standalone DHH/DHHA1 domain proteins have been identified in several bacteria including _B. burgdorferi_, _Mycobacterium_ spp., _Mycoplasma pneumoniae_, and _T. martiima_[87; 89; 90; 91; 92].  
Some DhhP enzymes show a preference for linear substrates over cyclic dinucleotides [95; 96]. Their essentiality varies across species; in _B. burgdorferi_ and _M. pneumoniae_, DhhP is the sole enzyme responsible for c-di-AMP degradation [87; 89]. Some homologs also function as nano-RNases, which degrade short RNA oligonucleotides [95]. [PERSON] et al. [87] found that, unlike in Gram-positive bacteria, DhhP is essential for _B. burgdorferi_ growth, and its conditional inactivation did not increase resistance to \(\beta\)-lactam antibiotics, suggesting that c-di-AMP functions may differ across bacterial phyla [87]. [PERSON] et al. further reported that DhhP is critical for potassium homeostasis in _Mycoplasma pneumoniae_, underscoring its role in bacterial adaptation [89]. The _dhhP_ mutant in _B. burgdorferi_ was defective in OspC production, a major virulence factor, and showed impaired mammalian infection, highlighting the potential of DhhP inhibitors as anti-virulence agents. Additionally, the mutant exhibited elongated cells, suggesting a role for DhhP in cell division (Figure 9B).  
[PERSON] et al. found that NrnA (also called Rv2847c or CdnP) inactivation in Mtb results in the accumulation of extracellular c-di-AMP, triggering a stronger inflammatory response from the host immune system. Due to its role in immune evasion, many research groups have shown interest in developing small-molecule inhibitors targeting NrnA. In 2021, the Sintm group reported **C82**[97], a selective, non-nucleotide-based NrnA inhibitor (Figure 9A). That same year, [PERSON] et al. patented a series of phosphonate-based NrnA inhibitors, including **I-7**[98]. More recently, in 2023, [PERSON] et al. identified another series of NrnA inhibitors, including **C-33** and **C-34**[99] (Figure 9A).  
Figure 8: (**A**) Domain structure of c-di-AMP phosphodiesterase [85; 86; 87; 93; 94]. (**B**) 3D crystal structure of Rv2837c bound to 5′-pApA (PDB:5J)U) [88].