Polyphosphate Kinase

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Introduction

Inorganic polyphosphate (polyP) is a linear P_i polymer linked by phosphoanhydride bonds. It exists in all cells in nature, and studies have shown that it may have been preserved from prebiotic times. The importance of polyP in bacteria has been fully confirmed. Under nutritional stringencies and environmental stresses, many Gram-negative bacteria will rapidly produce polyP. PolyP has multiple functions, which can regulate the expression of rpoS and recA at the transcriptional level, and affect the expression of many stress-inducible and stationary-phase-inducible genes. PolyP also regulates the degradation of ribosomal proteins through Lon protease.   

It is not clear how these deficiencies and stress signals lead to the accumulation of polyP. Polyphosphate kinase (PPK, also known as PPK1) is responsible for catalyzing the reversible conversion of the γ-phosphate of ATP to polyP. Deletion experiments of theppk gene in principal human pathogens (such as Pseudomonas aeruginosa) have demonstrated the importance of polyP and PPK in bacterial pathogenesis. PPK is also essential for motility, biofilm formation, and quorum sensing, which makes important contributions to the antibiotic resistance of pathogens. It is worth mentioning that the release of virulence factors also requires the participation of PPK. Since polyP is required for stationary-phase survival, PPK inhibitors may help eradicate non-multiplying bacteria. Currently, E. coliPPK has become a fully characterized enzyme. The purified recombinant PPK continues to catalyze the elongation of polyP and terminates the catalytic reaction when the polyP chain reaches about 750 phosphate groups.

Overall structure of Escherichia coli PPK

The crystal structure of the full-length E. coli PPK of both unliganded and AMPPNP-bound forms were determined in the same crystal lattice at 2.5 Å resolution. In the crystal lattice, each asymmetric unit contains two PPK monomers with basically the same structure, which are related by pseudo two-fold symmetry and form an interlocked dimer structure. When AMPPNP is combined with PPK, the conformation of the monomer only changes slightly. Each PPK monomer has an L-shaped structure with four domains. These domains include the amino-terminal domain, the "head" domain (H domain), and two closely related carboxy-terminal domains (C1 and C2 domains). The N domain includes residues 2-106, which consists of a bundle of three anti-parallel long α-helices located on the upper surface of the C-terminal domain. The N domain is a highly conserved region of PPK and provides the upper binding interface for the adenine ring of ATP. The C1 and C2 domains are composed of residues 322-502 and 503-687, respectively, and are highly conserved in the PPK family, the helix and β-sheet have different structural topologies and relative orientations Studies have shown that some residues that are essential for enzyme catalytic activity are located in these two domains.

Figure 1. Overall structure of Escherichia coli polyphosphate kinase (PPK) (Zhu, Y.; et al. 2015)

Chemical mechanism of PPK autophosphorylation

It has been shown that the autophosphorylation of PPK is the first step in polyp synthesis, and mutagenesis studies have shown that H435 and H454 may be the autophosphorylation site(s) of PPK. In the PPK-AMPPNP crystal structure, H435 directly interacts with the AMPPNP γ-phosphate group from the "reverse" position, while H454 is completely buried in the hydrophobic core of the C1 domain. This result provides evidence that H435 is the only autophosphorylation site of PPK. Some scientists speculate that H435 of PPK is used as a nucleophile to attack the phosphodiester bond of the γ-phosphate group of ATP, while H592 is used as a general acid protonating the oxygen atom between β- and γ-phosphate. There are four highly conserved amino acids that form key hydrogen bonds in the C1 and C2 domains: E623 with H435, D470 with H592. The possible role of D470 is to bind and correctly orientate the general acid H592. This model is consistent with the results of previous biochemical studies, that the mutants H435Q and H592Q failed to autophosphorylate.

Figure 2. The proposed chemical mechanism for PPK autophosphorylation (Zhu, Y.; et al. 2015)