## 3 PPK2 Enzymology
### PPK2 Crystal Structures and Catalytic Mechanism  
To date, the crystal structures of PPK2s from seven different bacterial species have been published (Table 1). These include representatives from all three classes: class I: _Sinorhizobium melitoli_ (PDB ID 3 CZQ) and _Francisella tulareis_ (PDB ID 4 YEG); class II: _P. aeruginosa_ PPK2C/PA3455 (PDB ID 3 CZP); and class III: _Arthobacter aurescens_ (PDB ID 3 RHF), _Meiothermus ruber_ (PDB ID 5 LC9), _Cytoplasa hutchinsonii_ (PDB ID 6 ANG), and _Deinococcus radiodurans_ (PDB ID 6 AQE) [36; 46; 52; 53]. These structures reveal a similar overall PPK2 architecture resembling that of thymidylate kinase [62]. Each PPK2 displays a three-layered \(\alpha\)-\(\beta\)-\(\alpha\) sandwich fold with a lid loop composed of two \(\alpha\)-helices. An Mg\({}^{2+}\) ion is frequently observed coordinated to the Walker A and/or Walker B motifs. The enzymes also share an internal substrate channel rich in positively charged residues that connect the polyP and nucleotide binding sites near the catalytic (Walker A) aspartate (Figure 2) [36; 46; 52; 53]. Uniquely, the class II _P. aeruginosa_ PPK2C has two fused PPK2 domains: a catalytically inactive N-terminal and an active C-terminal [36]. A comparison of the above crystal structures revealed a class-specific signature residue located after the Walker B motif: class I is asparagine (N153 in _S. melitoli_ PPK2), class II is glycine (G367 in PA3455), and class III is glutamate (E137 in _C. hutchinsonii_ PPK2) [46; 63]. Figure 2: PPK2 substrate binding sites elucidated via co-crystallization with polyP and nucleotide ligand. _F. tulareis_ PPK2 (green and purple) bound to polyP and the nucleotide AMP-PCH2 PPP (PDB ID 5 LLB) [53]. (**A**) Electrostatic surface representation of the polyP binding channel. Blue and red represent positive and negative charge, respectively. (**B**) Detailed view of the amino acid residues that mediate polyP binding. Interactions indicated by blue dashed lines. Figure created in Molecular Operating Environment. Several recent crystal structures of PPK2s complexed with polyP and/or nucleotides have provided invaluable insight into the substrate binding and reaction mechanisms of these enzymes. The class I _F. tulareis_ PPK2 (_Ft_PPK2) has been crystallized in complex with polyP alone (PDB ID 5 LL0) and both polyP and \(\beta\),\(\gamma\)-methylene adenosine 5\({}^{\prime}\)-pentaphosphate (AMP-PCPPP) (PDB ID 5 LLB). The structures of class III _M. ruber_ PPK2 (_Mr_PPK2) in complex with ADP and PPi (PDB ID 5 MAQ), AMP (PDB ID 5 LCD), ADP (PDB ID 5 LDB), and ATP (PDB ID 5 LD1) have also been solved. Likewise, class III _C. hutchinsonii_ PPK2 (_Ch_PPK2) in complex with AMP (PDB ID 6 ANG), ADP (PDB ID 6 AN9), and guanosine 5-tetraphosphate (G4P) (PDB ID 6 ANH) helps to further elucidate substrate specificity. _Ft_PPK2 co-crystallized with polyP revealed a polyP chain nine P\({}_{1}\) units long bound between the protein core and the lid loop. The polyP is positioned by a cluster of lysine and arginine residues, which are conserved across all PPK2s [53] and reminiscent of the polyP tunnel observed in PPK1 [37] (Figure 2A). _Ft_PPK2 co-crystallized with both polyP and the nucleotide \(\beta\),\(\gamma\)-methylene adenosine 5\({}^{\prime}\)-triphosphate (AMP-PCP) yielded a structure in which two additional phosphoryl units were transferred onto AMP-PCP, yielding AMP-PCPPP. The nucleotide was bound on the right side of the Walker B motif, as predicted by a prior [PERSON] et al. study [36], with its five phosphate groups extending leftward towards the polyP channel, guided via coordination to the lid loop Mg\({}^{2+}\) (Figure 2B). Although no electron density connecting the AMP-PCPPP to the polyP was visible, this spacing (\(\sim\)7.0 A) is consistent with the binding of six additional phosphoryl units. While intact polyP chains could not be observed in any of the _Mr_PPK2 structures, individual phosphate ions were observed in the region corresponding to the polyP channel in _Ft_PPK2.