## 3 PPK2 Enzymology  
A large body of phylogenetic, structural, and biochemical data exists for PPK2 enzymes. Sequence analysis suggests that PPK2s evolved from a common P-loop kinase  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Species** & **Number of PPK2s** & **PPK2 Class** & **PDB ID** & **Reference** \\ \hline _Acinetobacter baumaniii_ & 1 & II & [42] \\ \hline _Acinetobacter johnsonii_ & 1 & II & [43; 44] \\ \hline _Agrobacteriumium tunefaciens_ & 1 & I & [36; 45] \\ \hline _Arlrhobacter aurescens_ & 1 & III & 3 RHF & [46] \\ \hline _Bacillus cereus_ & 1 & II & [47] \\ \hline _Campylobacter jejuni_ & 1 & I & [48] \\ \hline _Corpheacterium glutamicum_ & 2 & I & [49] \\ \hline _Cytophaga hutchinsonii_ & 1 & III & 6 ANG, 6 ANH, 6 ANQ, 6 AUO, 6 AN9, 6B18 & [46] \\ \hline _Deinococcus geothermalis_ & 1 & III & [50] \\ \hline _Deinococcus radiothurans_ & 1 & III & 6 AQE, 7 NMJ, 7 BMM & [46; 50] \\ \hline _Delfia tsuruthentsis_ & 1 & III & [51] \\ \hline _Francisella tularensis_ & 1 & I & 4 YEG, 5 LLB, 5 LL0, 5 LLF & [52; 53] \\ \hline _Klebsiella pneumoniae_ & 1 & I & [42] \\ \hline _Meiothermus ruber_ & 1 & III & 5 LC9 & [50; 53] \\ \hline _Meiothermus silwanus_ & 1 & III & [50] \\ \hline _Mycobacterium singematis_ & 1 & I & [54] \\ \hline _Mycobacterium tuberculosis_ & 1 & I & [41; 55] \\ \hline _Myxococcus xanthus_ & 1 & II & [56] \\ \hline \multirow{6}{*}{_Pseudomonas aeruginosa_} & \multirow{3}{*}{3} & I (PPK2A/PA0141) & [34; 57] \\ \cline{3-3}  & & I (PPK2B/PA2428) & [39; 57] \\ \cline{3-3}  & & II (PPK2C/PA3455) & 3 CZP & [36] \\ \hline _Ralstonia eutropha_ & 5 & Not yet classified & [40; 58] \\ \hline _Rladopseudomonas pulstris_ & 1 & I & [36] \\ \hline _Ruegeria pomeroyi_ & 1 & I & [59] \\ \hline _Sinorhizobium melitoli_ & 3 & I & 3 CZQ, 6 DZG & [36] \\ \hline _Thermosynechococcus elongatus_ & 1 & III & [50] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Summary of bacterial species with characterized PPK2 enzymes.  
ancestor with thymidyllate kinases [36], which catalyze the phosphorylation of thymidine \(5^{\prime}\)-onophosphate by ATP to yield the essential DNA precursor thymidine \(5^{\prime}\)-diphosphate. P-loop kinases are characterized by a conserved Walker-A motif (GxxxGK) that interacts with the \(\beta\)-phosphate of the bound nucleotide, and a Walker B motif that mediates binding to the nucleotide \(\gamma\)-phosphate via Mg\({}^{2+}\) coordinated to a conserved carboxylate residue [60]. Phylogenetic analysis, combined with published biochemical data for select enzymes, revealed that PPK2s can be classified into three subfamilies based on nucleoside phosphate substrate preference: class I phosphorylate nucleoside diphosphates, class II phosphorylate nucleoside monophosphates, and class III can phosphorylate both nucleoside mono- or diphosphates [50] (Figure 1). Class III PPK2s, along with a common ancestor of class I and II PPK2, were likely the first to emerge, followed by diversification of class I and class II PPK2s. Since broad specificity is thought to be a property of primordial enzymes [61], the promiscuity of class III PPK2s supports the idea that this class is the closest to the original PPK2 ancestor [50].