# The HD-Domain Metalloprotein Superfamily:
### HD-Domain Hydrolases: The Phosphatase Subfamily
#### 1.2.3 Triphosphatases  
SAMHD1  
The sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a sophisticated deoxynucleotide triphosphate triphosphohydrolase that regulates dNTP levels in mammalian cells and plays a key role in HIV restriction, innate immunity, cancer, and cell cycle [2; 4; 46]. It does so by depleting cellular dNTP levels via an allosterically driven mechanism [47]. It has a strict specificity for deoxyribonucleotides (dGTP > dCTP > dTTP > dATP) [18] but can also hydrolyze other substrates to a lesser extent, including Clofarabine-TP, Ara-CTP, Ara-ATP, and Ara-UTP [48]. Hydrolysis of dNTPs by SAMHD1 is Mg-dependent and occurs with an apparent catalytic constant of 1-2 s\({}^{-1}\)[49], but the protein is also active with Mn. Optimal activity is contingent on the assembly of a homotetrameric complex that is afforded by the sequential binding of oxy and deoxyribonucleotides that act as allosteric activators [48]. SAMHD1 has two allosteric (AL) sites; AL-site 1 (D137, Q142, and R145) is tuned for binding both oxy and deoxy guanosine-based nucleotides, while AL-site 2 (N119, D330, N358, and R372) is selective for dNTPs [4]. The main catalytic core is held to the protein polypeptide by four invariant residues, H167, H206, D207, and D311, which until recently was well accepted to bind a single metal ion [18; 19]. A new structure, however, shows that the active site might be binuclear harboring a Fe-Mg center with the H233 coordinating the second metal ion (i.e., Mg) (Figure 3). The two metal ions are bridged by D207 and the substrate \(\alpha\)-phosphate [19]. Two histidine residues vicinal to the active site, H210 and H215, form salt bridges with the phosphate oxygens [18], and together with R164 and H233, are important for catalytic activity [18]. EF1143  
EF1143 from _Enterococcus faecalis_, a bacterium found in the human intestinal tract of humans, regulates the cellular levels of DNA building molecules by depleting the dNTP pools [20]. It is a distant homolog of SAMHD1 and shares some structural and functional similarities to the latter. Both SAMHD1 and EF1143 are homotetramers, and their first allosteric site is specific to guanosine-based triphosphates [4; 20; 48]. However, there are several salient differences between the two enzymes. EF1143 does not require dNTP binding for tetramerization, and in contrast to SAMHD1, the first allosteric site of EF1143 is strictly specific for dGTP [4; 20]. The active site is similar to that of SAMHD1, harboring residues H66, H110, D111, and D183 to coordinate a divalent metal ion. The Mg form of EF1143 is activated by dGTP and selectively hydrolyzes dATP and dCTP. In contrast, the Mn form does not require a nucleotide effector and hydrolyzes all dNTPs [20]. OxsA  
OxsA catalyzes the formation of the antitumor, antiviral, and antibacterial nucleoside analogue oxetancin-A (OXT) [22] (Figure 2). OxsA employs H31, H66, D67, and D132 to bind its metallocofactor for the sequential hydrolysis of phosphate groups from phosphorylated forms of OXT (i.e., OXT-P, OXT-PP, and OXT-PPP). The crystallographically observed cofactor nuclearity is dependent on the extent of substrate phosphorylation such that a mononuclear form catalyzes the hydrolysis of the singly phosphorylated OXT-P, while the binding of a second metal ion extends the hydrolytic capabilities to act on OXT-PP and OXT-PPP [22]. OxsA is most active with Co\({}^{2+}\) with apparent K\({}_{\text{cat}}\)/K\({}_{\text{M}}\) values of 106, 221, and 525 M\({}^{-1}\)s\({}^{-1}\) for OXT-P, OXT-PP, and OXT-PPP, respectively [22]. When OXT-P is bound, H66 occupies multiple orientations and can be replaced by water as a ligand to the metal ion. In contrast, when OXT-PP(P) is bound, H66 is a ligand to the active site metal. The second metal ion is octahedrally coordinated by D132, which bridges the two metal ions, water molecules, and the \(\beta\) and \(\gamma\) phosphate groups (Figure 3) [22].