## 2 PPK2: A New Class of PolyP-Metabolizing Enzyme  
While most of the early work on bacterial polyP used _E. coli_ (which encodes a single PPK1) as a model organism, other bacterial species such as _Pseudomonas aeruginosa_ were eventually examined. The Kornberg lab observed that their \(\Delta\)_ppK1_ knockout strain of _P. aeruginosa_ still produced up to 20% of wildtype polyP levels [33; 34], in contrast to \(\Delta\)_ppK1_\(E\). coli_ in which polyP is absent [6]. The source of this remaining polyP was later found to be a new class of enzyme termed PPK2 [34; 35]. Remarkably, PPK2 shares no significant sequence identity with PPK1 [35]. Subsequent X-ray crystal structures of PPK2s from several species also corroborated a distinct structural fold relative to that of PPK1 [36]. On a functional level, _P. aeruginosa_ PPK2 displayed a 75-fold preference for polyP degradation relative to synthesis, in contrast to PPK1 [34] (Figure 1). It therefore became clear that PPK1 and PPK2 were separate entities with distinct enzymology.  
Despite the absence of homology between PPK1 and PPK2, both respective classes of enzymes are highly conserved throughout the bacterial kingdom [35; 37]. Interestingly, it is postulated that PPK2 is evolutionarily older than PPK1 based on phylogenetic analysis and trends in amino acid loss and gain [38]. _ppk2_ genes have been identified in many species of both Gram-positive and Gram-negative bacteria [35], with several species encoding multiple distinct isoforms [35; 39; 40] (Table 1). For example, _P. aeruginosa_ possesses three distinct PPK2s [39], whereas other pathogenic bacteria such as _Mycobacterium tuberculosis_[41] or _Klebsiella pneumoniae_[42] possess only a single PPK2 isoform. In a more extreme instance, the environmental bacterium _Ralstonia eutropha_ contains five distinct PPK2 enzymes, further illustrating the genetic abundance and variability among PPK2s [40] (Table 1). By contrast, bacteria rarely encode more than one _ppk1_ allele [35; 40; 42].  
Figure 1: Comparison of the reactions catalyzed by PPK1 and PPK2 enzymes. R, nucleobase.  
The tendency of bacteria to encode one or more PPK2 isoform has in turn complicated the study of polyP physiology. _E. coli_ does not encode any PPK2s, and yet has served as the principal model species for studying bacterial polyP dynamics. As a result, our understanding of polyP dynamics in species other than _E. coli_ has suffered. For example, in _Acinetobacter baumaniii_--which is one of the few species known to encode multiple _ppk1_ genes in addition to _ppk2_--knockout of individual _ppk1s_ had differing consequences on virulence [42]. _P. aeruginosa_ possesses three PPK2s, only two of which, however, can synthesize polyP in vivo to compensate for loss of PPK1 [39; 57]. These studies challenge the validity of generalizing certain aspects of _E. coli_ polyP biology to organisms that possess PPK2s, since these enzymes may play species-specific niche roles. Furthermore, the presence of PPK2s in many of the current priority bacterial pathogens such as _P. aeruginosa_ and _A. baumaniii_ highlights the importance of understanding PPK2 biology in the fight against bacterial infections (Table 1) [42; 57].