## 2 Results  
### Hydrolytic Activity of hNudt16 on Dinucleotides Containing Diphosphate Bridge  
We have started our research by investigating the hydrolysis of diphosphate compounds. We were interested in whether GppG and AppA and their methylated counterparts are processed by hNudt16. We also performed measurements for previously published hNudt16 substrates, namely IDP, m\({}^{7}\)GDP, ADPr, dpCoA, NAD, and NADH. The hydrolysis reactions of all compounds were studied under the same experimental conditions (substrate concentration, buffer composition, and temperature). The products were analyzed and quantified using HPLC. Representative chromatograms for GppG are presented in Figure 1, and for m\({}^{7}\)GppG and AppA are shown in Figure S1. All investigated dinucleotides were hydrolyzed by hNudt16, while ADPr and m\({}^{7}\)GDP proved resistant to hydrolytic cleavage under the conditions tested (Table 1). The data presented in Table 1 indicate that unmethylated dinucleotide GppG was very efficiently hydrolyzed by hNudt16. The hydrolysis rate for GppG was much higher than that of its m\({}^{7}\)GppG counterpart. Dinucleotides containing adenine, AppA, dpCoA, NAD, and NADH were much less susceptible to enzymatic cleavage by this enzyme. These results are consistent with our previous report showing that dinucleotides containing adenine instead of guanine or methylated at the N7 position of guanine are inferior substrates for hNudt16 [12]. In the case of IDP, a very rapid hydrolysis reaction was observed. However, two factors made it impossible to correctly determine the hydrolysis rate: the very rapid progress of the reaction exceeding the capacity of the HPLC equipment and the formation of the IMP product, which had an inhibitory effect on the reaction. In brief, the results obtained from hydrolysis showed that hNudt16 has a high specificity towards IDP and GppG. The other compounds were hydrolyzed at several times slower reaction rates. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & & \multicolumn{2}{c}{**Hydrolysis Rate**} \\ \cline{3-4}
**Dinucleotide** & **Hydrolysis Products** & \multicolumn{2}{c}{**(\(\mu\)M/min/\(\mu\)g)**} & \multicolumn{2}{c}{**(\(\mu\)M/min/\(\mu\)E\({}_{0}\))**} \\ \hline GppG & GMP & 227.27 \(\pm\) 18.25 & 5454.5 \(\pm\) 436.3 \\ m\({}^{7}\)GppG & m\({}^{7}\)GMP + GMP & 30.11 \(\pm\) 2.81 & 722.6 \(\pm\) 68.6 \\ AppA & AMP & 1.70 \(\pm\) 0.12 & 40.8 \(\pm\) 2.9 \\ dpCoA & AMP + dpCop & 0.75 \(\pm\) 0.05 & 18.0 \(\pm\) 1.3 \\ NADH & AMP + NMNH & 0.78 \(\pm\) 0.06 & 18.7 \(\pm\) 1.5 \\ NAD & NMN + AMP & 0.14 \(\pm\) 0.02 & 3.4 \(\pm\) 0.4 \\ ADPr & nd & nd & \\ m\({}^{7}\)GDP & nd & nd & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Hydrolysis of dinucleotides catalyzed by hNudt16; nd—hydrolysis products not detected at conditions used for dinucleotides. Data are presented as v/\(\mu\)g (hydrolyzed substrate (\(\mu\)M) per minute) per enzyme (\(\mu\)g) and as v/\(\left|\text{E}\right|_{0}\) (hydrolyzed substrate (\(\mu\)M) per minute) per [enzyme] (\(\mu\)M). Figure 1: HPLC chromatograms for the hydrolysis of GppG catalyzed by hNudt16. (**A**) GppG in buffer (without hNudt16), (**B**) after 2 min reaction with hNudt16, (**C**) after 8 min reaction with hNudt16. Substrate concentration was 20 \(\mu\)M, enzyme concentration 0.002 \(\mu\)M. Chromatogram (**A**) was obtained after five-minute substrate incubation at 95 \({}^{\circ}\)C to show that heat inactivation of the enzyme in the assay buffer does not lead nonenzymatic degradation of GppG. Assessment of the Compliance of the Properties of hNudt16 Mutants with the Wild-Type Protein by Circular Dichroism (CD), Differential Scanning Fluorimetry (DSF)  
The problem encountered in quantifying the rate of IDP hydrolysis and the inability to compare GppG with hydrolysis product of known binding affinity as GMP or IMP prompted us to seek other methods to assess the Nudt16 specificity for studied compounds. Insights into this process can be gained by measuring the interaction of the protein with the ligand.