Inorganic polyphosphate functions at various stages of cell evolution

An important property of all chemical components of the living cells is their polyfunctionality.However, the complex of their functions may significantly change in the process of evolution. This can be well illustrated by the example of polyphosphates. In procaryotes, polyphosphates are involved in many biochemical and physiological processes and their metabolic regulation. Their metabolism is first closely connected with adenyl metabolism and bioenergetics. In the lower eucaryotes, the cells of which are evidently of endosymbiotic origin, polyphosphate metabolism of various organelles is considerably different and closely connected with the specificity of their function. In these organisms, polyphosphates are involved in metabolic and probably in genetic regulation of phosphate and adenyl metabolism. However, they first play the role of an osmotically inert reserve of inorganic phosphorus. In the higher animals having the hormonal and nervous systems of cell metabolism regulation, the function of polyphosphates as a metabolic regulator disappears. However, they apparently still function as regulators of gene expression and some transport processes.     

Glucose determination using immobilized polyphosphate glucokinase.

Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate:glucose phosphotransferase) was covalently coupled to collagen-coated silica gel beads. The immobilized enzyme, as a packed-bed reactor, was used to determine glucose in serum and other samples. The method was based on a spectrophotometric measurement of NADPH produced by two consecutive reactions, similar to the hexokinase method. The described approach takes advantage of the greater stability of polyphosphate compared to that of ATP, the greater specificity of polyphosphate glucokinase versus that of hexokinase, and the reusability of the immobilized enzyme. Linearity, precision, and accuracy of the method were tested and found to be very good. The results were linear between 10 and 50 nmol of glucose in a 50-microliter sample and the coefficient of variation was less than 4% in five successive determinations. The recovery of glucose was about 100% after calibration of the method. The results of the measurements correlated well with those obtained with soluble polyphosphate glucokinase (r = 0.997, y = 1.036x - 0.016). The immobilized-enzyme reactor showed good operational stability during a month of use, losing about 12% of its initial activity.     

The mechanism of utilization of polyphosphate by polyphosphate glucokinase from Propionibacterium shermanii

The phosphorylation of glucose by polyphosphate glucokinase with both long- and short-chain polyphosphates has been shown to occur by either a nonprocessive mechanism, i.e. with repeated association and dissociation of the polyphosphate from the enzyme after each phosphorylation or by a quasiprocessive mechanism in which several phosphorylations occur prior to the release of polyphosphate and the reassociation with the enzyme. In contrast, the phosphorylation of ADP to ATP by polyphosphate kinase is by a strictly processive mechanism; the phosphorylation occurs without release of the polymer from the enzyme prior to termination of the reaction (Robinson, N. A., Clark, J. E., and Wood, H. G. (1987) J. Biol. Chem. 262, 5216-5222). The demonstration that the mechanism is quasi-or nonprocessive was accomplished by electrophoresis using a variety of concentrations of polyacrylamide gels which made it possible to detect the intermediate sizes formed during the reactions. It also has been shown that all chains longer than about 100 are used simultaneously, but with chains of less than 100 residues, there is preferential utilization of the longest chains. Thus a narrow range of sizes is formed from a heterogeneous mixture of long chains. It is this formation of the narrow range of sizes that makes it possible to use polyphosphate glucokinase for the determination of the average size of long chains (Pepin, C. A., Wood, H. G., and Robinson, N. A. (1986) Biochem. Int. 12, 111-123).     

Role of ATP-glucokinase and polyphosphate glucokinase inStreptomyces aureofaciens

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomyceteStreptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC inStreptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.     

Influence of immobilization on properties of polyphosphate glucokinase

Polyphosphate glucokinase from Mycobacterium tuberculosis H37Ra was covalently bound to the glutaraldehyde-activated collagen coating silica gel, to the nylon, and to the CNBr-activated Sepharose. After immobilization kinetic properties of the enzyme were altered.     

Determination of the size of polyphosphates with polyphosphate glucokinase

A procedure for determining the size of inorganic polyphosphates of chain lengths up to about 750 is described. It involves reducing the size with polyphosphate glucokinase to a chain length that can be accurately determined by polyacrylamide gel electrophoresis. This measurement along with determination of the glucose-6-P formed and the total phosphate of the original polyphosphate permits calculation of the chain length. The accuracy of this method has been demonstrated by comparison with other reliable procedures. Thus far, it is the only method available for sizing long chain polyphosphates with nmol quantities.     

Inorganic polyphosphate and polyphosphate kinase: their novel biological functions and applications

In this review, we discuss the following two subjects: 1) the physiological function of polyphosphate (poly(P)) as a regulatory factor for gene expression in Escherichia coli, and 2) novel functions of E. coli polyphosphate kinase (PPK) and their applications. With regard to the first subject, it has been shown that E. coli cells in which yeast exopolyphosphatase (poly(P)ase), PPX1, was overproduced reduced resistance to H2O2 and heat shock as did a mutant whose polyphosphate kinase gene is disrupted. Sensitivity to H2O2 and heat shock evinced by cells that overproduce PPX1 is attributed to depressed levels of rpoS expression. Since rpoS is a central element in a regulatory network that governs the expression of stationary-phase-induced genes, poly(P) affects the expression of many genes through controlling rpoS expression. Furthermore, poly(P) is also involved in expression of other stress-inducible genes that are not directly regulated by rpoS. The second subject includes the application of novel functions of PPK for nucleoside triphosphate (NTP) regeneration. Recently E. coli PPK has been found to catalyze the kination of not only ADP but also other nucleoside diphosphates using poly(P) as a phospho-donor, yielding NTPs. This nucleoside diphosphate kinase-like activity of PPK was confirmed to be available for NTP regeneration essential for enzymatic oligosaccharide synthesis using the sugar nucleotide cycling method. PPK has also been found to express a poly(P):AMP phosphotransferase activity by coupling with adenylate kinase (ADK) in E. coli. The ATP-regeneration system consisting of ADK, PPK, and poly(P) was shown to be promising for practical utilization of poly(P) as ATP substitute.     

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP₇₅. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.     

A novel mammalian glucokinase exhibiting exclusive inorganic polyphosphate dependence in the cell nucleus

BackgroundHexokinase and glucokinase enzymes are ubiquitously expressed and use ATP and ADP as substrates in mammalian systems and a variety of polyphosphate substrates and/or ATP in some eukaryotic and microbial systems. Polyphosphate synthesising or utilizing enzymes are widely expressed in microbial systems but have not been reported in mammalian systems, despite the presence of polyphosphate in mammalian cells. Only two micro-organisms have previously been shown to express an enzyme that uses polyphosphate exclusively.MethodsA variety of experimental approaches, including NMR and NAD-linked assay systems were used to conduct a biochemical investigation of polyphosphate dependent glucokinase activity in mammalian tissues.ResultsA novel mammalian glucokinase, highly responsive to hexametaphosphate (HMP) but not ATP or ADP as a phosphoryl donor is present in the nuclei of mammalian hepatocytes. The liver enzyme exhibited sigmoidal kinetics with respect to glucose with a S_0.5 of 12 mM, similar to the known kinetics of mammalian ATP-glucokinase. The Km for HMP (0.5 mM) was also similar to that of phosphoryl donors for mammalian ATP-glucokinases. The new enzyme was inhibited by several nucleotide phosphates.ConclusionsWe report the discovery of a polyphosphate-dependent enzyme system in mammalian cells with kinetics similar to established ATP-dependent glucokinase, also known to have a nuclear location. The kinetics suggest possible regulatory or redox protective roles.General significanceThe role of polyphosphate in mammalian systems has remained an enigma for decades, and the present report describes progress on the significance of this compound in intracellular metabolism in mammals.     

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.     

Inorganic polyphosphates and heavy metal resistance in microorganisms

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO₄^? ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.

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Inorganic polyphosphate: toward making a forgotten polymer unforgettable.

Pursuit of the enzymes that make and degrade poly P has provided analytic reagents which confirm the ubiquity of poly P in microbes and animals and provide reliable means for measuring very low concentrations. Many distinctive functions appear likely for poly P, depending on its abundance, chain length, biologic source, and subcellular location. These include being an energy supply and ATP substitute, a reservoir for Pi, a chelator of metals, a buffer against alkali, a channel for DNA entry, a cell capsule and, of major interest, a regulator of responses to stresses and adjustments for survival in the stationary phase of culture growth and development. Whether microbe or human, we depend on adaptations in the stationary phase, which is really a dynamic phase of life. Much attention has been focused on the early and reproductive phases of organisms, which are rather brief intervals of rapid growth, but more concern needs to be given to the extensive period of maturity. Survival of microbial species depends on being able to manage in the stationary phase. In view of the universality and complexity of basic biochemical mechanisms, it would be surprising if some of the variety of poly P functions observed in microorganisms did not apply to aspects of human growth and development, such as aging and the aberrations of disease. Of theoretical interest regarding poly P is its antiquity in prebiotic evolution, which along with its high energy and phosphate content make it a plausible precursor to RNA, DNA, and proteins. Practical interest in poly P includes many industrial applications, among which is its use in the microbial depollution of P1 in marine environments.     

Effect of polyphosphate limitation on the anaerobic metabolism of phosphorus-accumulating microorganisms

There are two types of microbial populations described in the literature as being capable of anaerobic storage of acetic acid in activated-sludge processes: the polyphosphate-accumulating organisms (PAO) and the glycogen-accumulating non-polyphosphate organisms (GAO). Both groups use the conversion of glycogen to poly-hydroxyalkanoate to produce ATP and NADH; however, the first group can also produce ATP from polyphosphate (poly-P). No representative pure cultures are available from either group. The question arises: is the observed activity of GAO due to PAO that are depleted in poly-P?? In this study, using a laboratory sequencing batch reactor containing an enriched culture, the ability of the enriched PAO to utilize organic substrate (acetate) anaerobically was investigated under conditions of poly-P limitation and surplus glycogen content of the biomass. This study showed clearly that, under these conditions, almost no acetate was taken up. Furthermore, this strongly suggests that PAO can not use glycogen conversion to poly-hydroxyalkanoate as the sole energy source under anaerobic conditions, which seems to be the restricted to a separate group of GAO. On the basis of the results and literature data, an improved scheme for the anaerobic acetate accumulation is presented.     

Inorganic polyphosphate as an integral part of alkaline phosphatase preparations.

Alkaline phosphatase (from chicken intestinal sources) was shown to contain a considerable amount of polyanionic phosphorus which was released by basic digestion. The polyanionic phosphorus of alkaline phosphatase is not associated with protein or polyalcohols and does not exhibit a visible or ultraviolet absorption spectrum. Alkaline phosphatase and abiogenic inorganic polyphosphate were found to incorporate 32P-orthophosphate under similar experimental conditions. It has been previously reported that this enzyme will incorporate 32P-orthophosphate into its protein phosphoserine without the apparent concomitant utilization of an energy source. This reported phosphorylation was immediately reversible upon dilution of the phosphorylated enzyme with unlabelled orthophosphate, which indicates that the initial phosphorylation was an exchange reaction. These observations suggest that this polyanionic phosphorus from alkaline phosphatase may be inorganic polyphosphate.     

Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli.

The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth. After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium. The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl. After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored. Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase.