Fig. 4. NMR analysis of water-mediated SAMHD1 catalysis.

a Detailed view of the bi-metallic centre in the SAMHD1-active site. Metal ions and coordinated water molecules are shown as spheres, coloured by atom type. W0 is the activated water molecule positioned between the HD-bound Fe and Mg3, and orientated for nucleophilic attack on the α-phosphate. The electron density shown in blue mesh is the F_o − F_c map calculated during model building prior to inclusion of W0, contoured at 6 σ. b Chemical structures of the dGTP substrate and deoxyguanosine and triphosphate products of SAMHD1 hydrolysis, the α, β and γ-phosphate of dGTP together with the α*- and β*-phosphate of triphosphate are labelled. c, d SAMHD1 hydrolysis of dGTP monitored by ³¹P NMR. Spectra of dGTP substrate (left) and the triphosphate product (right) after 0 and 3 h incubation with SAMHD1 are shown. Assigned ³¹P resonances of the α-, β- and γ-phosphate of dGTP and α- and β-phosphate of the triphosphate product species are labelled. Product resonances are marked with asterisks. An expanded view of the α*-phosphate doublet resonance is shown inset. In c the reaction was carried out in solvent comprising 1:1 mixture of H₂O¹⁶ and H₂O¹⁸. The satellite peaks on α*-phosphate doublet resonance result from the O¹⁸ isotope effect, demonstrating that the reaction proceeds via a nucleophilic attack from a water molecule on the α-phosphate that is subsequently transferred into the triphosphate product.