# The HD-Domain Metalloprotein Superfamily:
### HD-Domain Hydrolases: The PDE Subfamily
#### 1.3.4 HD-Domain PDEs Acting on c-di-GMP and c-GAMP; the HD-GYP Subclass  
HD-GYPs are a special subclass of the PDE subfamily and are functionally homologous to EAL proteins (typified by the glutamate-alanine-leucine residue triad) [1; 58]. They can be single domain proteins or fusions to extra regulatory, sensory, or catalytic protein domains [8; 59; 60].  
While cyclic-di-GMP is their most common substrate, the recently discovered hybrid dinucleotide, 3'3'\(\mathrm{c}\)-GAMP, is also hydrolyzed by some HD-GYPs [59]. Out of the nine HD-GYPs encoded in _Vibrio cholerae_, VCA0681, VCA0931, and VCA0210 are the only HD-GYPs to hydrolyze both c-di-GMP and 3'3'\(\mathrm{c}\)-GAMP. More recently, PmxA from _Myxococcus xanthus_ was identified as a 3'3'\(\mathrm{c}\)-GAMP specific PDE that is hardly active toward c-di-GMP or c-di-AMP [60]. Selectivity for 3'3'\(\mathrm{c}\)-GAMP is attributedto a glutamine near the active site, although this residue is not conserved in VCA0681, VCA0931, and VCA0210, suggesting that the molecular origins for 3'3'cGAMP specificity may vary among HD-GYPs.  
In addition to the seventh ligand added to their active site (i.e., an extra histidine adjacent to the last histidine of the motif), all active HD-GYPs have a glycine-tyrosine-proline (GYP) residue triad in a loop close to the active site (Figure 5) [14; 35]. However, because single amino acid substitutions of each of the GYP domain residues to alanines hardly affect PDE activity [36], its role in catalysis and protein stability remains poorly understood. The GYP motif is considered important for interaction with the GGDEF cyclase (named after its highly conserved Gly-Gly-Asp-Glu-Phe sequence motif) [8] and serves as a substrate specificity element for the recognition of c-di-GMP and its hybrid 3'3'-cGAMP analog [40].  
HD-GYPs differ on the basis of their active metal cofactor and possible catalytic outcomes. While most commonly harbor a dimetal cofactor, some incorporate a trinuclear cofactor by involving a glutamate residue as an eighth ligand to the site (Figure 5) [36]. The assembly of a dinuclear or trinuclear cofactor is presumed to afford different reaction outcomes. Dinuclear HD-GYPs can only perform a one-step hydrolysis, whereas trinuclear ones can perform a two-step hydrolysis, leading to the respective monophophasties. Metal ions that can stimulate hydrolysis are Fe, Mn, and, to a lesser extent, Co and Ni [40].  
PmGH from _Persephonella marina_ is the prototypical trinuclear HD-GYP and the first to be crystallographically characterized [36]. PmGH harbors a triiron cofactor with the third iron coordinated by the glutamate E185. The trimetal cofactor is additionally stabilized by three other crystallization molecules, invoking the possibility that other solvent molecules may be incorporated under physiological conditions (Figure 5). It is active with both Fe\({}^{2+}\) and Mn\({}^{2+}\). On the basis of primary amino acid sequence, PA4781 from _Pseudomonas aeruginosa_ is also a putative trinuclear PDE; however, the available crystal structure shows two Ni ions in the active site at an elongated distance. PA4781 is unselective in its metal ion incorporation, has limited activity, and exhibits a preference for 5'-pGpG over c-di-GMP to form GMPs [14].  
Only one structure of a dinuclear HD-GYP exists: Bd1817 from _Bellowibrio bacteriovorus_. It harbors a diiron cofactor, but the presence of an asparagine instead of the last aspartate of the binding motif, a degraded GYP motif (Figure 5), and its complete inactivity toward c-di-GMP [35] do not allow for the inference of substrate positioning and specificity in dinuclear HD-GYPs.