Polyphosphate:AMP phosphotransferase as a polyphosphate-dependent nucleoside monophosphate kinase in Acinetobacter johnsonii 210A

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.     

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.     

Properties of polyphosphate: AMP phosphotransferase of Acinetobacter strain 210A.

Polyphosphate:AMP phosphotransferase, an enzyme which catalyzes the phosphorylation of AMP to ADP at the expense of polyphosphate, was purified more than 1,500-fold from Acinetobacter strain 210A by streptomycin sulfate precipitation and by Mono-Q, Phenyl Superose, and Superose column chromatography. Streptomycin sulfate precipitation appeared to be an effective step in the purification procedure. During the following chromatographic steps, there was a 29-fold increase in specific activity but the yield was low (0.3%). Kinetic studies showed apparent Km values of 0.26 mM for AMP and 0.8 microM for polyphosphate with an average chain length of 35 phosphate groups. The highest activities were found with polyphosphate molecules of 18 to 44 phosphate residues. The polyphosphate chain was degraded completely to ADP. The mechanism of degradation is processive. No activity was obtained with ortho-, pyro-, tri-, and tetraphosphate. The enzyme was inhibited by pyro-, tri-, and tetraphosphate. The inhibition by tri- and tetraphosphate was mixed with polyphosphate as a substrate. The inhibition constants for the dissociation of the enzyme-inhibitor complex and for the enzyme-inhibitor-substrate complex were 0.9 and 6.5 mM, respectively, for triphosphate and 0.7 and 1.5 mM, respectively, for tetraphosphate.     

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.     

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.     

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg² was required foractivity, the activity with Mn², Co² and Zn² was 48, 48 and 182% of the activity observed with Mg², respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent K_m for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.     

ATP production from polyphosphate in Acinetobacter strain 210A

In cell-free extracts of Acinetobacter strain 210A polyphosphate: AMP phosphotransferase and adenylate kinase activity was measured. Polyphosphate glucokinase and polyphosphate dependent NAD kinase were not detected. The specific activity of polyphosphate: AMP phosphotransferase was found to be 43 nmol · min^-1 · mg^-1 protein in presence of 1 mmol · l^-1 AMP. The adenylate kinase reaction had an equilibrium constant ([ATP] [AMP] [ADP]^-2) of 0.7, an activity of 54 nmol · min^-1 · mg^-1 protein, and was almost completely inhibited by 0.3 mM P¹,P⁵-di(adenosine-5)-pentaphosphate. ATP was formed through the combined action of polyphosphate: AMP phosphotransferase and adenylate kinase in cell-free extracts from bacterial polyphosphate and from chemically prepared polyphosphate (Graham's salt). A spectrophotometric method for the continuous monitoring of polyphosphate: AMP phosphotransferase is also presented.Abbreviations Ap₅A P¹,P⁵-di(adenosine-5)-pentaphosphate - G6P-DH D-glucose-6-phosphate dehydrogenase - HK hexokinase - AEC adenylate energy charge - U units (converting 1 mol · min^-1)     

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.     

The elemental composition dynamics of large polyphosphate granules in Acinetobacter strain 210A

Electron microscopy and energy dispersive X-ray micro-analysis were used to examine the elemental composition of large polyphosphate granules in unfixed and unstained intact cells of Acinetobacter strain 210A. When grown in medium with butyrate, Acinetobacter strain 210A possessed 1 or 2 large granules with a diameter of 0.4 m besides a relatively large number of small granules. The large granules were composed of phosphorus, magnesium and potassium. A decrease in the Mg/Ca-ratio of the medium from 5.95 to 0.0073 resulted in a decline in the intracellular Mg/Ca-ratio from 15 to 0.56. At a high intracellular Mg/Ca-ratio, magnesium was the dominant counterion in the polyphosphate granule. Calcium became the major cation in the polyphosphate bodies at a low intracellular Mg/Ca-ratio. Omission of Ca²⁺ or modification of the K/Mg ratio in the medium did not significantly affect the cation composition of the polyphosphate granules. The dissociation constants for Mg- and Ca-polyphosphate were 9.3×10^-2 mol/l and 1.5×10^-1 mol/l, respectively.     

Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1     

A novel beta-diketone-cleaving enzyme from Acinetobacter johnsonii: acetylacetone 2,3-oxygenase

A novel Fe+Zn containing oxygenase from Acinetobacter johnsonii catalyses 2,3-cleavage of acetylacetone to acetate and methylglyoxal has been purified. The stoichiometry of reactants and products conforms to a classical dioxygenase. The pure protein is a homotetramer of 64kD with variable amounts of Fe(2+) and Zn(2+). Activity of the enzyme is more closely related to the Fe(2+) content than to the amount of protein. A purification of acetylacetone 2,3-oxygenase, some of its physical properties, and the preference for some analogous substrates are described.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

Polyphosphate production by strains of Acinetobacter

Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA3123. No correlation was found between the polyphosphate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.     

Characterization of restriction endonuclease AjoI from Acinetobacter johnsonii

Abstract A new type II restriction endonuclease, named Ajo I, was detected in Acinetobacter johnsonii . The enzyme Ajo I, an isoschizomer of Pst I, recognized the hexanucleotide sequence [5'-CTGCA/G-3'], with a cleavage site generating fragments of DNA with protruding cohesive 3' termini.     

Hydrogenosomal ATP:AMP phosphotransferase of Trichomonas vaginalis

1. ATP:AMP phosphotransferase (adenylate kinase) is present in Trichomonas vaginalis, primarily with hydrogenosomal localization. 2. Adenylate kinase has been purified from hydrogenosome-enriched fractions by solubilization with Triton X-100 and KCl followed by affinity chromatography and gel filtration. 3. The enzyme has a Mr = 28,000, a broad pH optimum of pH 7-9, requirement for Mg2+ and specificity for adenine and deoxyadenine nucleotides. 4. The activity is competetively inhibited by P1,P5-di(adenosine-5') pentaphosphate (Ki 200 nM) and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoate). 5. Catalytic properties of this enzyme are similar to those of enzymes from other organisms. Other properties indicate its uniqueness, however, since its molecular mass and Ki for P1,P5-di(adenosine-5'-)-pentaphosphate bring it closer to the mitochrondrial isoenzyme, while it shares a requirement for reduced thiol groups with the cytosolic isoenzyme.     

Intracellular localization of ATP:AMP phosphotransferase in Escherichia coli

ATP and AMP were immediately converted into ADP by intact cells of Escherichia coli in the presence of Mg2+, while ADP was also rapidly converted into ATP and AMP under the same conditions. Adenylate kinase was released when E. coli cells were converted to spheroplasts by treatment with lysozyme-EDTA or osmotic shock. Adenylate kinase activities detected in the cytoplasm, periplasm and membrane fractions were approximately 58%, 36% and 6% of the total cellular activity, respectively. These results indicate that adenylate kinase in E. coli occurs in the periplasm as well as the cytoplasm.     

ATP:AMP phosphotransferase activity, a new characteristic of Catharanthus roseus tonoplasts

The ATPase activity of Catharanthus roseus tonoplasts was examined using HPLC separation and quantification of adenine nucleotides. ATP seemed to be degraded into ADP and AMP by tonoplast vesicles. When ADP was the initial substrate, the appearance of AMP and concomitant ATP synthesis were observed; these reactions were inhibited by Ap⁵A. The apparent degradation of ATP into AMP was also inhibited by Ap⁵A. These results indicated that AMP arose from an ATP:AMP phosphotransferase activity and excluded the possibility of the hydrolysis of ADP into AMP by the tonoplast ATPase. AMP was degraded by the microsomal fraction from protoplasts or by the cytosol while the tonoplast vesicles did not hydrolyze it. This observation was used to assess the purity of tonoplasts.