Polyphosphate Synthetic Activity of Polyphosphate:AMP Phosphotransferase in Acinetobacter johnsonii 210A

AMP formation by PAP.

To examine the reversibility of the PAP reaction, we monitored the dephosphorylation of ADP by detecting AMP formation under the optimum Mg²⁺ concentration (100 mM) of the PAP forward reaction at 37°C, as shown in Fig. 1. The AMP and ADP concentrations in the reaction mixture were then measured by using an high-pressure liquid chromatography system equipped with a TSK gel ODS-120T column (Tosoh, Tokyo, Japan) at 25°C. The mobile phase was 30 mM diethylaminoethanol, 20 mM phosphate, and 2% (wt/vol) methanol in water, which was delivered at a flow rate of 1.0 ml min⁻¹. The eluent was monitored by UV detection at 260 nm. In the presence of both AMP (5 mM) and poly(P) (20 mM, in terms of phosphate residues) in the reaction mixture, 93.9% of AMP was converted to ADP by the forward reaction of PAP. On the other hand, when only ADP (5 mM) was added in the reaction mixture, 5.1% of ADP was converted to AMP. These results suggest that both reactions reached the equilibrium state in the phosphate conversion between AMP and ADP and that PAP catalyzed the reverse reaction (presented above), which produced poly(P) from ADP. The equilibrium ratio for the AMP and ADP formation by PAP was 1:19 in the presence of 100 mM MgCl₂ and was changed to 1:9.6 (9.4% of ADP [5 mM] was converted to AMP) in the presence of 20 mM MgCl₂ (Fig. 1). Therefore, we examined poly(P)-synthetic activity of PAP with 20 mM MgCl₂ in the reaction mixture.

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FIG. 1.

Equilibration of reactions for poly(P) synthesis and utilization by PAP. For the poly(P) utilization reaction, the reaction mixture (500 μl) contained 50 mM Tris-HCl (pH 8.0), 20 mM poly(P), 5 mM AMP, and 0.12 U of purified PAP (dimer fraction) with 100 mM (○) or 20 mM (□) MgCl₂. For the poly(P) synthetic reaction, the reaction mixture (500 μl) contained 50 mM Tris-HCl (pH 8.0), 5 mM ADP, and 0.12 U of purified PAP (dimer fraction) with 100 mM (•) or 20 mM (▪) MgCl₂. Reactions were performed at 37°C.

Poly(P) synthesis by reverse reaction of PAP.

In order to prove that PAP has poly(P)-synthetic activity, the product of the reverse reaction was analyzed by gel electrophoresis. ADP at 100 mM was incubated at 37°C with purified PAP (4.6 U ml⁻¹) in the presence of 20 mM MgCl₂, and the resultant reaction products were also analyzed by using polyacrylamide gel electrophoresis (Fig. 2A). Then, 10 μl of the reaction mixtures was loaded with 5 μl of loading dye solution (40 mM Tris-HCl buffer [pH 8.3], 1 mM EDTA, 20% glycerol, 0.05% bromophenol blue) on a 15% polyacrylamide gel (270 mm high by 165 mm wide and 1.0 mm thick). Next, 50 μg of standard poly(P)s (sodium phosphate glass type 65 [average chain length is ca. 61; Sigma] and sodium phosphate glass type 75+ [average chain length is ca. 84; Sigma]) were loaded as poly(P) size markers. Poly(P) synthesized by purified E. coli PPK (average chain length is ca. 750) (1) was also loaded as another size reference. Gel was stained by a 0.05% toluidine blue containing 0.5% glycerol for 5 min and was then destained by water. Compared to the poly(P) synthesized by E. coli PPK, PAP catalyzes the synthesis of a shorter poly(P) ranging from 50 to 700 residues. The average chain length of poly(P) synthesized by PAP was ca. 200 to 500. Since almost all of the products of PAP were concentrated at 200 to 500 poly(P) chain lengths, poly(P) synthesis by PAP seems to be processive.

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FIG. 2.

Poly(P) synthesis from ADP by PAP reverse reaction. (A) Gel electrophoresis (5, 17) of poly(P) synthesized by PAP. After incubation of the reaction mixture for the indicated times (20, 60, and 120 min), the products (PAP product) were separated by 15% polyacrylamide gel electrophoresis. Poly(P) type 65 (P65) at 50 μg, poly(P) type P75+ (P75) at 50 μg, and poly(P) synthesized by purified E. coli PPK (P750) were used as size markers. (B) Amounts of AMP (○) and poly(P) (•) synthesized by PAP in the reaction mixture. To determine poly(P) concentration synthesized by PAP, the reaction mixtures described above were hydrolyzed by rPPX1 by incubating 0 to 30 mM poly(P) (reaction products of PAP) (30 μl) with 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, and 1.7 mU of purified rPPX1 at 37°C for 1 h. The amounts of released inorganic phosphate were estimated by the method of Chen et al. (4). Poly(P) concentrations are presented in terms of phosphate residues.

To further confirm poly(P) synthetic activity, the amount of poly(P) synthesized by PAP was estimated by using the determination of released inorganic phosphate after the hydrolysis of the poly(P) by yeast recombinant exopolyphosphatase (rPPX1) (17) (Fig. 2B). The released orthophosphate concentration was measured by using the method of Chen et al. (4). At the same time, the AMP and ADP concentrations in the same PAP reaction mixture were also measured by using an high-pressure liquid chromatography system equipped with a TSK gel ODS-120T column. Figure 2B shows the amount of AMP and poly(P) in the reaction mixture. The orthophosphate concentration released from poly(P) by rPPX1 treatment is in agreement with the increase of AMP concentration. This suggests that poly(P) synthesis depends on the dephosphorylation of ADP resulting in the formation of AMP.

Optimizations of MgCl₂ concentration and pH for AMP formation by PAP.

To determine the optimum concentration of MgCl₂ for dephosphorylation of ADP, we evaluated the reverse reaction of PAP by measuring AMP formation at various concentrations of MgCl₂. The highest activity for AMP formation was observed in 20 mM MgCl₂, whereas the optimum MgCl₂ concentration for poly(P) degradation (ADP synthesis) was 100 mM (Shiba et al., unpublished) (Fig. 3). No precipitation was observed in the reaction mixture containing up to 100 mM MgCl₂ in the presence of 10 mM poly(P), although other metal ions such as Mn, Co, and Fe caused precipitation with 10 mM poly(P) even at lower concentrations (<20 mM). This indicates that Mg could be a preferable metal ion that enhances PAP activity without causing of precipitation and that Mn cannot replace Mg for the activation of PAP activity (Shiba et al., unpublished). We confirmed the Mg specific enhancement of the poly(P)-utilization activity by replacing Mg with Mn and found that only 7.9% of maximum activity was detected when the optimum concentration of Mn (5 mM) was added to the reaction mixture (Shiba et al., unpublished).

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FIG. 3.

Effect of MgCl₂ concentrations on poly(P) synthetic and utilization activity of PAP. Reaction mixtures (50 μl each) containing purified PAP, 50 mM Tris-HCl (pH 9.0), and 5 to 100 mM MgCl₂ were incubated at 37°C with 5 mM ADP for the poly(P) synthetic reaction (○) and with 5 mM AMP and 10 mM poly(P) for the poly(P) utilization reaction (•). The values for the activity are presented as relative values in which the activity with 20 mM MgCl₂ for the poly(P) synthetic activity and 100 mM MgCl₂ for the poly(P) utilization activity are set at 100%, respectively.

One hypothesis is that the equilibrium ratio between ADP and AMP formation was changed by MgCl₂ concentration in the reaction mixture, as shown in Fig. 1. This means that Mg²⁺ may play a crucial role in controlling the balance between poly(P) synthesis and utilization. A. johnsonii 210A was isolated as a strain that can accumulate up to 300 mg of poly(P) per g (dry weight) (16). When the energy generation is repressed (as, for example, in the absence of oxygen or an electron donor), poly(P) is degraded and inorganic phosphate is released into the medium (15, 16). In such energy-starved conditions, as in the stationary phase, the concentration of free Mg²⁺ available for the PAP reaction might be different from that in log-phase cells. Furthermore, for poly(P) utilization, we also observed maximum activity in 20 mM MgCl₂, together with 50 mM NH₄SO₄ in the reaction mixture (Shiba et al., unpublished), whereas no enhancement of poly(P) synthetic activity was observed upon the addition of NH₄SO₄ (data not shown). The alterations of not only the Mg concentration but also combinations of Mg and other salt concentrations may be able to control the poly(P) level in the cells depending on the equilibrium ratio of the PAP reaction.

We examined the optimum pH for the reverse reaction of PAP by using 50 mM concentration of maleate buffer (pH 5.0 to 7.0), Tris-HCl buffer (pH 7.0 to 9.0), and glycine buffer (pH 9.0 to 11.0) by monitoring the ADP hydrolysis activity of PAP (data not shown). We found that the optimum pH for the PAP reverse reaction was 9.0, which was in the same range as for the forward reaction (8.0 to 9.0) (Shiba et al., unpublished).

Comparison of poly(P) synthetic activities among PAP, PPK1, and PPK2.

We compared the characteristics of PAP for poly(P) synthesis with other poly(P) kinases, PPK1 from E. coli (1, 2, 12) and PPK2 from P. aeruginosa (9, 18), and the results are summarized in Table 1. The C-terminal region of PAP (amino acid numbers 236 to 475 in GenBank accession no. AB092983 ) has a 60.5% identity with the similar region of PPK2 (amino acid number 71-256 in GenBank accession no. AY168003 ) in P. aeruginosa, even though the two proteins are only 15.8% identical when the entire sequences are aligned. PAP is different from other PPKs because the PAP synthesizes poly(P) by using the nucleoside “di”-phosphate and not by using the “tri”-phosphate. As substrates, PPK1 can only use ATP, but PPK2 uses both ATP and GTP (9, 18). In the case of PAP, the poly(P) synthetic activity from GDP (5 mM) was also observed, but the level was only 1.1% of that from ADP (5 mM) (data not shown). The kinetic parameters of PAP for poly(P) synthesis from ADP were also determined by the Lineweaver-Burk plot. The K_m value of PAP for ADP is 8.3 mM. The V _max value of PAP for poly(P) synthetic activity is 55 μmol min⁻¹ mg⁻¹, which is 1.1- and 7.2-fold greater than those for PPK1 and PPK2, respectively. PAP and PPK2 have strong poly(P) utilization activity (V _max), and the activity of PAP is 20-fold higher than that in PPK1, even though the K_m values for utilization of all three enzymes are almost the same. This implies that PAP and PPK2 work mainly for poly(P) utilization in the cell. However, PAP has the same level of poly(P) synthetic activity (K_m and V _max) as PPK1, and this suggests that PAP also works for poly(P) synthesis at a significant level. Combining these results, we can conclude that PAP itself has the ability to work both in poly(P) utilization and synthesis, whereas PPK1 and PPK2 work mainly for poly(P) synthesis and utilization, respectively. To determine the reaction direction (synthesis or utilization) of PAP, intracellular concentrations of ADP, AMP, and poly(P), as well as Mg and other salt concentrations might be critical.

Although there is no report about the existence of ppk1 in the A. johnsonii genome, Zhang et al. (18) reported the existence of ppk1 in the genomes of Acinetobacter spp., including Acinetobacter baumannii, A. calcoaceticus, and Acinetobacter sp. ADP1. Thus, we suppose that A. johnsonii also has ppk1. PAP and PPK1 may function in poly(P) synthesis depending on the condition. For example, PPK1 can synthesize poly(P) in energy-rich aerobic growth condition, whereas PAP may work in ADP-rich conditions in which the energy state is not preferable for accumulating ATP. This is consistent with PAP is high K_m value for ADP (8.3 mM). PAP can degrade poly(P) under low-energy conditions (i.e., stationary phase or anaerobic condition) when excess AMP and poly(P) accumulates.

One possibility is that A. johnsonii has an efficient phosphate recycling system involving poly(P), AMP, ADP, and ATP. PAP could contribute to this recycling system by making poly(P) from ADP, in addition to making ADP from poly(P) and AMP. The poly(P) utilization (ADP synthetic) activity of PAP in combination with ADK also makes it possible to synthesize ATP from AMP. PAP might therefore be a key enzyme for intracellular phosphate recycling in A. johnsonii 210A.