The multiple activities of polyphosphate kinase of Escherichia coli and their subunit structure determined by radiation target analysis

Polyphosphate kinase (PPK), the principal enzyme required for the synthesis of inorganic polyphosphate (polyP) from ATP, also exhibits other enzymatic activities, which differ significantly in their biochemical optima and responses to chemical agents. These several activities include: polyP synthesis (forward reaction), nATP --> polyP(n) + nADP (Equation 1); ATP synthesis from polyP (reverse reaction), ADP + polyP(n) --> ATP + polyP(n - 1) (Equation 2); general nucleoside-diphosphate kinase, GDP + polyP(n) --> GTP + polyP(n - 1) (Equation 3); linear guanosine 5'-tetraphosphate (ppppG) synthesis, GDP + polyP(n) --> ppppG + polyP(n - 2) (Equation 4); and autophosphorylation, PPK + ATP --> PPK-P + ADP (Equation 5). The Mg(2+) optima are 5, 2, 1, and 0.2 mM, respectively, for the activities in Equations 1, 2, 3, and 4. Inorganic pyrophosphate inhibits the activities in Equations 1 and 3 but stimulates that in Equation 4. The kinetics of the activities in Equations 1, 2, and 3 are highly processive, whereas the transfer of a pyrophosphoryl group from polyP to GDP (Equation 4) is distributive and demonstrates a rapid equilibrium, random Bi-Bi catalytic mechanism. Radiation target analysis revealed that the principal functional unit of the homotetrameric PPK is a dimer. Exceptions are a trimer for the synthesis of ppppG (Equation 4) and a tetrameric state for the autophosphorylation of PPK (Equation 5) at low ATP concentrations. Thus, the diverse functions of this enzyme involve different subunit organizations and conformations. The highly conserved homology of PPK among 18 microorganisms was used to determine important residues and conserved regions by alanine substitution, by site-directed mutagenesis, and by deletion mutagenesis. Of 46 single-site mutants, seven exhibit none of the five enzymatic activities; in one mutant, ATP synthesis from polyP is reduced relative to GTP synthesis. Among deletion mutants, some lost all five PPK activities, but others retained partial activity for some reactions but not for others.     

Molecular analysis of polyphosphate accumulation in bacteria

The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed.     

The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein.

Polyphosphate kinase (PPK) catalyzes the reversible transfer of the terminal phosphate of ATP to form a long-chain polyphosphate (polyP) (Ahn, K., and Kornberg, A. (1990) J. Biol. Chem. 265, 11734-11739). The Escherichia coli gene (ppk) encoding PPK has been cloned, sequenced, and overexpressed (about 100-fold). The gene possesses an open reading frame for 687 amino acids (mass of 80,278 Da). PPK has been purified from overproducing cells after release from attachment to the cell outer membrane; the purified soluble PPK reassociate with cell membrane fractions. About 850 molecules of PPK are found in a wild type cell.     

Inorganic polyphosphate is required for motility of bacterial pathogens

The ppk gene encodes polyphosphate kinase (PPK), the principal enzyme in many bacteria responsible for the synthesis of inorganic polyphosphate (polyP) from ATP. A null mutation in the ppk gene of six bacterial pathogens renders them greatly impaired in motility on semisolid agar plates; this defect can be corrected by the introduction of ppk gene in trans. In view of the fact that the motility of pathogens is essential to invade and establish systemic infections in host cells, this impairment in motility suggests a crucial and essential role of PPK or polyP in bacterial pathogenesis.     

多聚磷酸相关蛋白结构及生物学功能

多聚磷酸（polyphosphate,polyP）是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶（polyphosphate kinase,PPK）催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶（exopolyphosphatase,PPX）、内切聚磷酸酶（endopolyphosphatase,PPN）能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD）,使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate,NADP）.多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.     

Polyphosphate kinase of Acinetobacter sp. strain ADP1: purification and characterization of the enzyme and its role during changes in extracellular phosphate levels.

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl(2). The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40 degrees C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per microg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P(i)) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P(i)-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P(i) conditions. This activity declined when phosphate was added.     

Crystallization and characterization of polyphosphate kinase from Escherichia coli

Linear polyphosphate chains have been found to play a key role in bacterial responses to stresses and nutritional depletion, and are necessary for host infection of various pathogens. Polyphosphate kinase (PPK) is a critical enzyme responsible for polyphosphate synthesis in bacteria. PPK knockout mutations in several Gram-negative pathogens identify PPK as an ideal drug target for the development of a new class of antibacterial drugs. To reveal the catalytic mechanism and provide a structural basis for drug discovery, we have purified and crystallized full-length Escherichia coli PPK and its complex with AMP-PNP. The crystals diffract to a resolution of 2.5A and belong to the space group P4(2)2(1)2 with unit-cell parameters a=152.0, b=152.0, and c=150.0 A. Crystal structure of PPK is being determined by the Se-Met MAD experiment.     

The glycogen-bound polyphosphate kinase from Sulfolobus acidocaldarius is actually a glycogen synthase

Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya, Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162-5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.     

Polyphosphate Kinase of Acinetobacter sp. Strain ADP1: Purification and Characterization of the Enzyme and Its Role during Changes in Extracellular Phosphate Levels

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl₂. The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40°C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per μg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P_i) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P_i-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P_i conditions. This activity declined when phosphate was added.     

Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2

Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzyme's catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.     

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported.     

Discovery of potent polyphosphate kinase 1 (PPK1) inhibitors using structure‐based exploration of PPK1Pharmacophoric space coupled with docking analyses

Inorganic polyphosphate (polyP) is present in all living forms of life. Studied mainly in prokaryotes, polyP and its associated enzymes are vital in diverse metabolic activities, in some structural functions, and most importantly in stress responses. Bacterial species, including many pathogens, encode a homolog of a major polyP synthesis enzyme, Poly Phosphate Kinase (PPK) with 2 different genes coding for PPK1 and PPK2. Genetic deletion of the ppk1 gene leads to reduced polyP levels and the consequent loss of virulence and stress adaptation responses. This far, no PPK1 homolog has been identified in higher‐order eukaryotes, and, therefore, PPK1 represents a novel target for chemotherapy. The aim of the current study is to investigate PPK1 from Escherichia coli with comprehensive understanding of the enzyme's structure and binding sites, which were used to design pharmacophores and screen a library of compounds for potential discovery of selective PPK1 inhibitors. Verification of the resultant inhibitors activities was conducted using a combination of mutagenic and chemical biological approaches. The metabolic phenotypic maps of the wild type E. coli (WT) and ppk1 knockout mutant were generated and compared with the metabolic map of the chemically inhibited WT. In addition, biofilm formation ability was measured in WT, ppk1 knockout mutant, and the chemically inhibited WT. The results demonstrated that chemical inhibition of PPK1, with the designed inhibitors, was equivalent to gene deletion in altering specific metabolic pathways, changing the metabolic fingerprint, and suppressing the ability of E. coli to form a biofilm.     

The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.     

Magnetic resonance-based visualization of gene expression in mammalian cells using a bacterial polyphosphate kinase reporter gene

Gene expression reporter systems, in which a promoter of interest is cloned upstream of a readily assayed reporter gene, have been developed and used extensively to study gene expression in prokaryotes and eukaryotes. Unfortunately, most of these systems cannot be used to assay gene expression in nonsuperficial tissues in living organisms. This study examines a novel reporter gene system based on the gene encoding Escherichia coli polyphosphate kinase (PPK), which can be used to monitor gene expression in mammalian cells. PPK catalyzes the synthesis of inorganic polyphosphate (polyP) from ATP, and because mammalian cells do not contain detectable levels of polyP, PPK activity can be measured in mammalian cells using 31P-magnetic resonance spectroscopy or 31P-magnetic resonance imaging. The ppk reporter gene system described here is noninvasive, does not require an exogenous substrate, and can potentially be used in internal tissues of living organisms.     

Different chain length specificity among three polyphosphate quantification methods

Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbuscular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which polyphosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples.     

Influence of nitrogen and phosphorus on polyphosphate accumulation in Gigaspora margarita during spore germination

Polyphosphate (polyP) is the form in which phosphorus (P) is transferred from extraradical hyphae into arbuscles in the symbiotic stage of arbuscular mycorrhizal fungi. However, polyP dynamics in the presymbiotic stage are less understood. In this study, we aimed to investigate polyP accumulation in Gigaspora margarita as influenced by nitrogen (N) and/or P supply during germination. Spores of G. margarita were incubated on medium with or without P or N addition. PolyP content in the fungal tissue was monitored using a polyP kinase/luciferase system, and polyP synthetic activity was determined with ³²P labeling. The results showed that both N and P were necessary for polyP accumulation in germ tubes. Nitrate increased the polyP content in germ tubes, but ammonium did not. Along with germination, polyP content decreased in spores, but increased in germ tubes. ³²P labeling indicated that polyP synthetic activity increased in germ tubes along with germination, but was negligible in spores. Our results suggest that, in the presymbiotic stage of G. margarita, uptake of environmental N and P increases polyP content in germ tubes, and that polyP synthesis occurs mainly therein, leading to polyP accumulation. The possible mechanism of transfer of polyP from spores to hyphae remains to be elucidated.     

PPK1 and PPK2 — which polyphosphate kinase is older?

Polyphosphate kinases (PPKs) catalyse the polymerisation and degradation of polyphosphate chains. As a result of this process, PPK produces or consumes energy in the form of ATP. Polyphosphate is a linear molecule that contains tens to hundreds of phosphate residues connected by macroergic bonds, and it appears to be an easily obtainable and rich source of energy from prebiotic times to the present. Notably, polyphosphate is present in the cells of all three domains of life, but PPKs are widely distributed only in Bacteria, as Archaea and Eucarya use various unrelated or “nonhomologous” proteins for energy and metabolic balance. The present study focuses on PPK1 and PPK2 homologues, which have been described to some extent in Bacteria, and the aim was to determine which homologue group, PPK1 or PPK2, is older. Phylogenetic analyses of 109 sequence homologues of Escherichia coli PPK1 and 109 sequence homologues of Pseudomonas aeruginosa PPK2 from 109 bacterial genomes imply that polyphosphate consumption (PPK2) evolved first and that phosphate polymerisation (PPK1) evolved later. Independently, a theory of the trends in amino acid loss and gain also confirms that PPK2 is older than PPK1. According to the results of this study, we propose 68 hypothetical proteins to mark as PPK2 homologues and 3 hypothetical proteins to mark as PPK1 homologues.     

in Silico analysis of Escherichia coli polyphosphate kinase (PPK) as a novel antimicrobial drug target and its high throughput virtual screening against PubChem library

Multiple drug resistance (MDR) in bacteria is a global health challenge that needs urgent attention. The 2011 outbreak caused by Escherichia coli O104:H4 in Europe has exposed the inability of present antibiotic arsenal to tackle the problem of antimicrobial infections. It has further posed a tremendous burden on entire pharmaceutical industry to find novel drugs and/or drug targets. Polyphosphate kinase (PPK) in bacteria plays a crucial role in helping latter to adapt to stringent conditions of low nutritional availability thus making it a good target for antibacterials. In spite of this critical role, to best of our knowledge no in-silico work has been carried out to develop PPK as an antibiotic target. In the present study, virtual screening of PPK was carried out against all the 3D compounds with pharmacological action present in PubChem database. Our screening results were further refined by interaction maps to eliminate the false positive data respectively. From our results, compound number 5281927 (PubChem ID) has been found to have significant affinity towards affinity towards PPK active ATP-binding site indicating its therapeutic relevance.