Publications

• Origin of exopolyphosphatase processivity: fusion of an ASKHA phosphotransferase and a cyclic nucleotide phosphodiesterase homolog.

  Alvarado J., Ghosh A., Janovitz T., Jauregui A., Hasson M.S., Sanders D.A.

  The Escherichia coli Ppx protein is an exopolyphosphatase that degrades long-chain polyphosphates in a highly processive reaction. It also hydrolyzes the terminal 5' phosphate of the modified nucleotide guanosine 5' triphosphate 3' diphosphate (pppGpp). The structure of Ppx has been determined to ... >> More

  The Escherichia coli Ppx protein is an exopolyphosphatase that degrades long-chain polyphosphates in a highly processive reaction. It also hydrolyzes the terminal 5' phosphate of the modified nucleotide guanosine 5' triphosphate 3' diphosphate (pppGpp). The structure of Ppx has been determined to 1.9 A resolution by X-ray crystallography. The exopolyphosphatase is an ASKHA (acetate and sugar kinases, Hsp70, actin) phosphotransferase with an active site found in a cleft between the two amino-terminal domains. Analysis of the active site indicates that among the ASKHA phosphotranferases of known structure, Ppx is the closest to the ectonucleoside triphosphate diphosphohydrolases. A third domain forms a six-helix claw that is similar to the catalytic core of the eukaryotic cyclic nucleotide phosphodiesterases. Most of the 29 sulfate ions bound to the Ppx dimer occupy sites where the polyP chain likely binds. An aqueduct that passes through the enzyme provides a physical basis for the enzyme's high processivity. << Less

  Structure 14:1263-1272(2006) [PubMed] [EuropePMC]

• The role of the novel exopolyphosphatase MT0516 in Mycobacterium tuberculosis drug tolerance and persistence.

  Thayil S.M., Morrison N., Schechter N., Rubin H., Karakousis P.C.

  Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been charac ... >> More

  Inorganic polyphosphate (poly P) has been postulated to play a regulatory role in the transition to bacterial persistence. In bacteria, poly P balance in the cell is maintained by the hydrolysis activity of the exopolyphosphatase PPX. However, the Mycobacterium tuberculosis PPX has not been characterized previously. Here we show that recombinant MT0516 hydrolyzes poly P, and an MT0516-deficient M. tuberculosis mutant exhibits elevated intracellular levels of poly P and increased expression of the genes mprB, sigE, and rel relative to the isogenic wild-type strain, indicating poly P-mediated signaling. Deficiency of MT0516 resulted in decelerated growth during logarithmic-phase in axenic cultures, and tolerance to the cell wall-active drug isoniazid. The MT0516-deficient mutant showed a significant survival defect in activated human macrophages and reduced persistence in the lungs of guinea pigs. We conclude that exopolyphosphatase is required for long-term survival of M. tuberculosis in necrotic lung lesions. << Less

  PLoS ONE 6:E28076-E28076(2011) [PubMed] [EuropePMC]

• Pseudomonas aeruginosa exopolyphosphatase is also a polyphosphate: ADP phosphotransferase.

  Beassoni P.R., Gallarato L.A., Boetsch C., Garrido M.N., Lisa A.T.

  Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn-1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing ... >> More

  Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn-1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4 (+) is an activator of the enzyme and may function at concentrations lower than those of K(+); (iii) Zn(2+) is also an activator of paPpx and may substitute Mg(2+) in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg(2+) and capable of producing ATP regardless of the presence or absence of K(+) or NH4 (+) ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity. << Less

  Enzyme Res. 2015:404607-404607(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The gene for an exopolyphosphatase of Pseudomonas aeruginosa.

  Miyake T., Shiba T., Kameda A., Ihara Y., Munekata M., Ishige K., Noguchi T.

  In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The ... >> More

  In Pseudomonas aeriginosa, a gene, ppx, that encodes exopolyphosphatase [exopoly(P)ase; EC 3.6.1.11] of 506 amino acids (56,419 Da) was found downstream of the gene for polyphosphate kinase, ppk. Since ppx is located in the opposite direction of the ppk gene, they do not constitute an operon. The predicted amino acid sequence of PPX is 41% identical with Escherichia coli PPX. The gene product of ppx (paPPX) was overproduced in E. coli, and its activity was evaluated. Orthophosphate (Pi) is released from polyphosphate [poly(P)], the average chain lengths of which are 79 and 750, respectively. The amount of Pi released matched the amount of poly(P) lost. Thus ppx encodes an enzyme that has exopoly(P)ase activity. << Less

  DNA Res. 6:103-108(1999) [PubMed] [EuropePMC]

• Endopolyphosphatase in Saccharomyces cerevisiae undergoes post-translational activations to produce short-chain polyphosphates.

  Shi X., Kornberg A.

  Endopolyphosphatase (Ppn), responsible for cleavage of long chain inorganic polyphosphate (poly P) of several hundred residues to generate progressively shorter chains, has been identified in mammalian cells and purified from Saccharomyces cerevisiae. Disruption of the encoding gene, PHM5, in S. c ... >> More

  Endopolyphosphatase (Ppn), responsible for cleavage of long chain inorganic polyphosphate (poly P) of several hundred residues to generate progressively shorter chains, has been identified in mammalian cells and purified from Saccharomyces cerevisiae. Disruption of the encoding gene, PHM5, in S. cerevisiae resulted in a mutant that showed limited growth and failure to survive in a minimal medium. The limited digestion products of the yeast enzyme Ppn1 judged to be P(3) and P(60) have now, with the homogeneous enzyme and improved separation methods, been demonstrated to be P(i) and P(3). Ppn1, a homotetramer of a 35-kDa subunit, is of vacuolar origin and requires protease activation of a 78 kDa (674-aa) precursor polypeptide (prePpn1). The protease-processed Ppn1 has been purified 3800-fold to homogeneity and the protease cleavage sites determined. Both termini of prePpn1 and the post-translational modification of N-glycosylations are essential for the protease-mediated maturation of Ppn1. << Less

  FEBS Lett. 579:2014-2018(2005) [PubMed] [EuropePMC]

• Deficiency of the novel exopolyphosphatase Rv1026/PPX2 leads to metabolic downshift and altered cell wall permeability in Mycobacterium tuberculosis.

  Chuang Y.M., Bandyopadhyay N., Rifat D., Rubin H., Bader J.S., Karakousis P.C.

  <h4>Unlabelled</h4>Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key ... >> More

  <h4>Unlabelled</h4>Mycobacterium tuberculosis can persist for decades in the human host. Stringent response pathways involving inorganic polyphosphate [poly(P)], which is synthesized and hydrolyzed by polyphosphate kinase (PPK) and exopolyphosphatase (PPX), respectively, are believed to play a key regulatory role in bacterial persistence. We show here that M. tuberculosis poly(P) accumulation is temporally linked to bacillary growth restriction. We also identify M. tuberculosis Rv1026 as a novel exopolyphosphatase with hydrolytic activity against long-chain poly(P). Using a tetracycline-inducible expression system to knock down expression of Rv1026 (ppx2), we found that M. tuberculosis poly(P) accumulation leads to slowed growth and reduced susceptibility to isoniazid, increased resistance to heat and acid pH, and enhanced intracellular survival during macrophage infection. By transmission electron microscopy, the ppx2 knockdown strain exhibited increased cell wall thickness, which was associated with reduced cell wall permeability to hydrophilic drugs rather than induction of drug efflux pumps or altered biofilm formation relative to the empty vector control. Transcriptomic and metabolomic analysis revealed a metabolic downshift of the ppx2 knockdown characterized by reduced transcription and translation and a downshift of glycerol-3-phosphate levels. In summary, poly(P) plays an important role in M. tuberculosis growth restriction and metabolic downshift and contributes to antibiotic tolerance through altered cell wall permeability.<h4>Importance</h4>The stringent response, involving the regulatory molecules inorganic polyphosphate [poly(P)] and (p)ppGpp, is believed to mediate Mycobacterium tuberculosis persistence. In this study, we identified a novel enzyme (Rv1026, PPX2) responsible for hydrolyzing long-chain poly(P). A genetically engineered M. tuberculosis strain deficient in the ppx2 gene showed increased poly(P) levels, which were associated with early bacterial growth arrest and reduced susceptibility to the first-line drug isoniazid, as well as increased bacterial survival during exposure to stress conditions and within macrophages. Relative to the control strain, the mutant showed increased thickness of the cell wall and reduced drug permeability. Global gene expression and metabolite analysis revealed reduced expression of the transcriptional and translational machinery and a shift in carbon source utilization. In summary, regulation of the poly(P) balance is critical for persister formation in M. tuberculosis. << Less

  MBio 6:E02428-E02428(2015) [PubMed] [EuropePMC]

• An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon.

  Akiyama M., Crooke E., Kornberg A.

  A gene, ppx, that encodes a novel exopolyphosphatase of 513 amino acids (58,133 Da) was found downstream of the gene for polyphosphate kinase, ppk. Transcription of the ppx gene depends on the ppk promoters, indicating a polyphosphate (polyP) operon of ppk and ppx. Exopolyphosphatase, purified to ... >> More

  A gene, ppx, that encodes a novel exopolyphosphatase of 513 amino acids (58,133 Da) was found downstream of the gene for polyphosphate kinase, ppk. Transcription of the ppx gene depends on the ppk promoters, indicating a polyphosphate (polyP) operon of ppk and ppx. Exopolyphosphatase, purified to homogeneity from overproducing cells, is judged to be a dimer of 58-kDa subunits. Orthophosphate is released processively from the ends of polyP approximately 500 residues long, but chains of approximately 15 residues compete poorly with polyP as substrate; ATP is not a substrate. Mg2+ (1 mM) and a high concentration of K+ (175 mM) support optimal activity. << Less

  J. Biol. Chem. 268:633-639(1993) [PubMed] [EuropePMC]

• Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell.

  Van Dien S.J., Keyhani S., Yang C., Keasling J.D.

  The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids. The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter. Polyphosphatase, a poly ... >> More

  The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids. The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter. Polyphosphatase, a polyphosphate depolymerase, was similarly expressed by using the arabinose-inducible PBAD promoter. The ability of cells containing these constructs to produce active enzymes only when induced was confirmed by polyphosphate extraction, enzyme assays, and RNA analysis. The inducer concentrations giving optimal expression of each enzyme were determined. Experiments were performed in which ppk was induced early in growth, overproducing PPK and allowing large amounts of polyphosphate to accumulate (80 mumol in phosphate monomer units per g of dry cell weight). The ppx gene was subsequently induced, and polyphosphate was degraded to inorganic phosphate. Approximately half of this polyphosphate was depleted in 210 min. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells and was secreted into the medium, leading to a down-regulation of the phosphate-starvation response. In addition, the steady-state polyphosphate level was precisely controlled by manipulating the degree of ppx induction. The polyphosphate content varied from 98 to 12 mumol in phosphate monomer units per g of dry cell weight as the arabinose concentration was increased from 0 to 0.02% by weight. << Less

  Appl. Environ. Microbiol. 63:1689-1695(1997) [PubMed] [EuropePMC]