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.     

In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2'-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl(2), polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

In Vitro ATP Regeneration from Polyphosphate and AMP by Polyphosphate:AMP Phosphotransferase and Adenylate Kinase from Acinetobacter johnsonii 210A

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP_n + AMP → ADP + polyP_n−1. The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2′-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2′-dAMP were efficiently phosphorylated to ADP and 2′-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl₂, polyP_n=35, and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2′-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.     

多聚磷酸激酶及其在ATP再生体系构建中的进展

构建高效的腺嘌呤核苷三磷酸(adenosinetriphosphate,ATP)再生体系可显著提高生物催化磷酸基团转移反应的效率。多聚磷酸激酶(poly phosphate kinase, PPK)能利用来源广、廉价且稳定的多聚磷酸(polyphosphate, Poly P)盐作为磷酸基供体，能够实现单磷酸腺苷(adenosine monophosphate,AMP)、二磷酸腺苷(adenosinediphosphate,ADP)、ATP、PolyP之间磷酸基的高效定向转移，已成为构建ATP再生体系的首选。本文介绍了不同类型PPK的结构特征、相关催化机制以及不同来源的PPK在酶活、催化效率、稳定性和底物偏好性的特征差异；归纳和列举了针对野生PPK酶学性质不足进行分子改造的实例，并对PPK在ATP再生体系构建的研究进展进行了总结。     

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.     

ATP amplification for ultrasensitive bioluminescence assay: detection of a single bacterial cell

We developed an ultrasensitive bioluminescence assay of ATP by employing (i) adenylate kinase (ADK) for converting AMP + ATP to two molecules of ADP, (ii) polyphosphate (polyP) kinase (PPK) for converting ADP back to ATP (ATP amplification), and (iii) a commercially available firefly luciferase. A highly purified PPK-ADK fusion protein efficiently amplified ATP, resulting in high levels of bioluminescence in the firefly luciferase reaction. The present method, which was approximately 10,000-fold more sensitive to ATP than the conventional bioluminescence assay, allowed us to detect bacterial contamination as low as one colony-forming unit (CFU) of Escherichia coli per assay.     

Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.     

Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase

1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.     

Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A

Polyphosphate:AMP phosphotransferase (PAP) has been identified as an enzyme that catalyzes the phosphorylation of AMP with inorganic polyphosphates [poly(P)] as phosphate donors. We found that the purified PAP of Acinetobacter johnsonii 210A has poly(P) synthetic activity. The PAP catalyzes the dephosphorylation of ADP and processively synthesizes poly(P) of 200 to 700 residues. Comparatively lower concentrations of MgCl(2) (20 mM) were required to obtain optimum poly(P) synthetic activity, whereas higher concentrations of MgCl(2) (100 mM) were necessary for optimum PAP activity. ADP is preferred over GDP as a phosphate donor for poly(P) synthesis. The K(m) and V(max) values for ADP in the poly(P) synthetic activity of PAP were 8.3 mM and 55 micromol min(-1) mg(-1), respectively. We concluded that the PAP of A. johnsonii 210A is a novel type of poly(P) kinase that uses ADP and GDP as substrates.     

Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate

Polyphosphate kinase (PPK) polymerizes the terminal phosphate of ATP to a long chain polyphosphate (poly(P) or (Pi)n) in a freely reversible reaction (Kornberg, S. R. (1957) Biochim. Biophys. Acta 26, 294-300), nATP in equilibrium nADP + (Pi)n, PPK, now purified to homogeneity, is a tetramer of 69-kDa subunits. Addition of a primer in the synthetic reaction is not required, nor does ATP or inorganic orthophosphate (Pi) serve in this role. PPK is autophosphorylated under the conditions of poly(P) synthesis; Pi is linked by a nitrogen-phosphate bond as judged by its acid lability and alkali stability. Incorporation of phosphate from the isolated phosphoenzyme into poly(P) upon the addition of ATP in the synthetic reaction and its incorporation into ATP upon the addition of ADP indicate phosphoenzyme to be an intermediate in the reaction. At an ATP level of 5 microM, well below its Km of 2 mM, a pronounced lag in poly(P) synthesis can be removed by tetrapolyphosphate but not by Pi, PPi, or tripolyphosphate. The basis for this stimulatory effect is not clear inasmuch as tetrapolyphosphate does not promote the dephosphorylation of the presumed phosphoenzyme intermediate.     

Immobilized polyphosphate kinase: preparation, properties, and potential for use in adenosine 5'-triphosphate regeneration

Polyphosphate kinase (ATP:polyphosphate phosphotransferase; EC 2.7.4.1), partially purified from Escherichia coli, has been immobilized on glutaraldehyde-activated aminoethyl cellulose with a 10% retention of enzymatic activity. The immobilized enzyme can carry out the synthesis of ATP from ADP, using long-chain inorganic polyphosphate as a phosphoryl donor. Chromatographic analyses of the product mixture produced from ADP and [32P]polyphosphate demonstrated that 98% of the 32P was incorporated into ATP, indicating that the immobilized polyphosphate kinase is substantially free from contaminating polyphosphate phosphohydrolase (EC 3.6.1.11), adenosine triphosphatase (EC 3.6.1.4), and adenylate kinase (EC 2.7.4.3). Immobilized polyphosphate kinase loses no activity when stored in an aqueous suspension for 2 months at 5 degrees C or for 1-2 weeks at 25 degrees C. It may be stored indefinitely as a lyophilized powder at -10 degrees C. Michaelis constants for ADP and polyphosphate were determined to be 160 and 120 microM, respectively, for the immobilized enzyme. A small-batch reactor was found to produce ATP linearly with time up to 65% conversion of polyphosphate into ATP and to attain greater than 85% conversion to ATP at equilibrium. The ease of purification and immobilization of E. coli polyphosphate kinase, its storage stability, the purity and yield of its ATP product, and the low values of the Michaelis constants for its substrates make it a highly promising enzyme for ATP regeneration.     

Polyphosphate:AMP phosphotransferase and polyphosphate:ADP phosphotransferase activities of Pseudomonas aeruginosa

In Pseudomonas aeruginosa PAO1, we have found massive polyphosphate:AMP phosphotransferase activity and polyphosphate:ADP phosphotransferase activity known as the reverse catalytic activity of polyphosphate kinase which participates in polyphosphate synthesis in the bacterium. Biochemical analysis using the partially purified polyphosphate:ADP phosphotransferase has revealed that it is independent of polyphosphate kinase and can function as polyphosphate-dependent nucleoside diphosphate kinase which most prefers GDP to the other three nucleoside diphosphates as a phospho-acceptor. It has been also demonstrated that polyphosphate:AMP phosphotransferase activity marked in the bacterium mainly originates from the combined action of the polyphosphate:ADP phosphotransferase described above and adenylate kinase. Both of the polyphosphate-utilizing activities require short polyP as a phospho-donor whose chain length is <75.     

Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.     

幽门螺杆菌PPK 的纯化与功能分析*

目的:表达纯化幽门螺杆菌多聚磷酸激酶,并测定其功能。方法:将幽门螺杆菌多聚磷酸激酶基因克隆入原核表达载体PQE80L中,在大肠杆菌(E.coli)DH5-α中表达。用BD Talon resin纯化目的蛋白。并在体外测定其合成多聚磷酸盐及转化多聚磷酸盐至ATP的能力。结果:成功构建了原核表达载体,得到高表达量的融合蛋白。经BD Talon resin纯化获得较高纯度的His-多聚磷酸激酶N端融合蛋白。体外实验证实该酶可以有效合成不同链长的多聚磷酸盐,并且在适当条件下可将多聚磷酸盐转化为ATP。结论:利用原核表达载体可很好表达幽门螺杆菌多聚磷酸激酶,纯化后的蛋白具有良好生物活性,是一个具备合成多聚磷酸盐及转化其为ATP的双向功能的酶。     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum

Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with (31)P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.     

利用ATP扩增反应与生物发光法结合检测微量微生物

摘要:【目的】腺苷酸激酶（adenylate kinase, ADK）和多聚磷酸盐激酶（polyphosphate kinase, PPK）偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇（Polyurethane）的磁珠（magnetic beads）,通过化学反应将腺苷酸双磷酸酶（apyrase）固定于磁珠表面,制备固相腺苷酸双磷酸酶（Beads-apyrase）,用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。     

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.     

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.