|
| |
Journal of Bacteriology, August 2004, p . 5178-5181, Vol . 186,
No . 15
Polyphosphate Synthetic Activity of Polyphosphate:AMP Phosphotransferase in
Acinetobacter johnsonii 210A
Hiromichi Itoh and Toshikazu Shiba*
Frontier Research Division, Fujirebio, Inc., Hachioji, Tokyo 192-0031, Japan
Received 16 January 2004/ Accepted 5 May 2004
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 MgCl2 (20 mM) were required
to obtain optimum poly(P) synthetic activity, whereas higher
concentrations of MgCl2 (100 mM) were necessary for optimum
PAP activity . ADP is preferred over GDP as a phosphate donor
for poly(P) synthesis . The Km and Vmax
values for ADP in the poly(P) synthetic activity of PAP were 8.3 mM
and 55 µmol 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 (PPK; EC 2.7.4.1) and polyphosphate:AMP
phosphotransferase (PAP) are enzymes that can catalyze the phosphorylation
of nucleotides by using inorganic polyphosphates [poly(P)] as
phosphate donors (1-3, 6-13,
18; T . Shiba, H . Itoh, A . Kameda, K . Kobayashi, Y .
Kawazoe, and T . Noguchi, unpublished data; T . Shiba and A . Kornberg,
unpublished data) . PPK can transfer the terminal phosphate residue of
poly(P) to ADP to yield ATP . PPK also catalyzes the processive
synthesis of poly(P) from ATP . Two types of PPKs, PPK1 and PPK2, have
been identified and are widely found in the bacterial genomes (18) .
PPK1, which was first isolated from Escherichia coli, prefers
poly(P) synthesis to poly(P) utilization . In contrast, PPK2, which
was recently identified in Pseudomonas aeruginosa PAO1,
prefers poly(P) utilization, resulting in nucleotide triphosphate
formation (9, 18) . PPK1 and PPK2
have no similarity in their amino acid sequences and were also
distinguished with biochemical characteristics such as kinetics,
ADP/GDP selectivity as substrates, requirement of metal ions, and
their active conformations (9, 18) .
PAP catalyzes the conversion of the terminal phosphate residue of
poly(P) to AMP, resulting in the synthesis of ADP . Its activity was
first discovered in Corynebacterium xerosis (6), and
the enzyme was partially purified and characterized in
Acinetobacter johnsonii 210A (3) . Its marked
activity was also found in Myxococcus xanthus, and the
activity in the membrane fraction was characterized (10;
Shiba and Kornberg, unpublished) . Weak PAP activity was detected in
E . coli, although this activity may be the consequence of the
combined action of PPK and adenylate kinase (ADK) (7) .
Recently, we cloned the pap gene from A . johnsonii 210A and
found that the amino acid sequence of PAP (GenBank accession
no.
AB092983) was highly homologous with those of PPK2 (GenBank
accession no.
AY168003) and its homologues (GenBank accession no.
NP_251118 and
NP_252145) isolated from P . aeruginosa PAO1 (Shiba et al.,
unpublished) .
It has been incorrectly considered that PAP has only a poly(P)-utilizing
activity (forward reaction) because the poly(P) synthetic activity
of PAP in both the crude extract and partially purified PAP
enzyme had not been reported (3, 14) . Thus,
the PAP reaction has been believed to be irreversible (3) .
However, discoveries of extensive homology between PAP and PPK2
encourage us to consider the possibility that PAP has poly(P)
synthetic activity as its reverse reaction . The forward and reverse
reactions were as follows: poly(P)n + AMP
poly(P)n – 1 + ADP . In order to
investigate whether or not PAP catalyzes the poly(P) synthetic
reaction, we evaluated the reverse reaction of PAP by using a highly
purified PAP enzyme .
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 .
|
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 .
|
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) .
|
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 Km
value of PAP for ADP is 8.3 mM . The Vmax 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 (Vmax),
and the activity of PAP is 20-fold higher than that in PPK1, even
though the Km 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 (Km and Vmax)
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 .
| TABLE 1 . Comparison of poly(P) synthetic and utilization activities of
PAP, PPK1, and PPK2
|
|
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
Km 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 .
We thank T . Noguchi (YAMASA corporation) for sending highly purified
GDP . We also thank A . Kornberg and K . Ishige for valuable
suggestions .
This study was supported by a Grant-in-Aid for the Creation of
Innovations through the Business-Academic-Public Sector Cooperation
and a Grant-in-Aid for Scientific Research on Priority Areas from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan .
* Corresponding author . Mailing address: Frontier Research
Division, Fujirebio, Inc., Hachioji, Tokyo 192-0031, Japan . Phone:
81-426-45-4755 . Fax: 81-426-45-4755 . E-mail: shiba@regenetiss.com .
- Ahn, K., and A . Kornberg. 1990 . Polyphosphate kinase from
Escherichia coli: purification and demonstration of a phosphoenzyme
intermediate . J . Biol . Chem . 265:11734-11739 .
- Akiyama, M., E . Crooke, and A . Kornberg. 1992 . The
polyphosphate kinase gene of Escherichia coli: isolation and sequence
of the ppk gene and membrane location of the protein . J . Biol . Chem .
267:22556-22561 .
- Bonting, C . F., G . J . Kortstee, and A . J . Zehnder. 1991 .
Properties of polyphosphate:AMP phosphotransferase of Acinetobacter
strain 210A . J . Bacteriol . 173:6484-6488.
- Chen, P . S., Jr., T . Y . Toribara, and H . Warner. 1956 .
Microdetermination of phosphorus . Anal . Chem . 28:1756-1758.
- Clark, J . E., and H . G . Wood. 1987 . Preparation of
standards and determination of sizes of long-chain polyphosphates by gel
electrophoresis . Anal . Biochem . 161:280-290.
- Dirheimer, G., and J . P . Ebel. 1965 . Caractérisation
d'une polyphosphate-AMP-phosphotransférase dans Corynebacterium xerosis .
C . R . Acad . Sci . 260:3787-3790.
- Ishige, K., and T . Noguchi. 2000 . Inorganic polyphosphate
kinase and adenylate kinase participate in the polyphosphate:AMP
phosphotransferase activity of Escherichia coli . Proc . Natl . Acad . Sci .
USA 97:14168-14171 .
- Ishige, K., and T . Noguchi. 2001 . Polyphosphate:AMP
phosphotransferase and polyphosphate:ADP phosphotransferase activities of
Pseudomonas aeruginosa . Biochem . Biophys . Res . Commun . 281:821-826.
- Ishige, K., H . Zhang, and A . Kornberg. 2002 .
Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP .
Proc . Natl . Acad . Sci . USA 99:16684-16688 .
- Kameda, A., T . Shiba, Y . Kawazoe, Y . Satoh, Y . Ihara, K .
Ishige, and T . Noguchi. 2001 . A novel ATP regeneration system using
polyphosphate-AMP phosphotransferase and polyphosphate kinase . J . Biosci .
Bioeng . 91:557-563.
- Kornberg, S . R. 1957 . Adenosine triphosphate synthesis
from polyphosphate by an enzyme from Escherichia coli . Biochim .
Biophys . Acta 26:294-300.
- Kuroda, A., and A . Kornberg. 1997 . Polyphosphate kinase
as a nucleoside diphosphate kinase in Escherichia coli and
Pseudomonas aeruginosa . Proc . Natl . Acad . Sci . USA 94:439-442 .
- Ogawa, N., C.-M . Tzeng, C . D . Fraley, and A . Kornberg.
2000 . Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical,
and physiologic features . J . Bacteriol . 182:6687-6693 .
- Van Groenestijn, J . W., M . M . Bentvelsen, M . H . Deinema, and
A . J . Zehnder. 1989 . Polyphosphate-degrading enzymes in Acinetobacter
spp . and activated sludge . Appl . Environ . Microbiol . 55:219-223.
- Van Groenestijn, J . W., M . H . Deinema, and A . J . Zehnder.
1987 . ATP production from polyphosphate in Acinetobacter strain 210A .
Arch . Microbiol . 148:14-19.
- Van Groenestijn, J . W., M . Zuidema, J . J . van de Worp, M . H .
Deinema, and A . J . Zehnder. 1989 . Influence of environmental parameters on
polyphosphate accumulation in Acinetobacter sp . Antonie Leeuwenhoek
55:67-82.
- Wurst, H., T . Shiba, and A . Kornberg. 1995 . The gene for
a major exopolyphosphatase of Saccharomyces cerevisiae . J . Bacteriol.
177:898-906.
- Zhang, H., K . Ishige, and A . Kornberg. 2002 . A
polyphosphate kinase (PPK2) widely conserved in bacteria . Proc . Natl . Acad .
Sci . USA 99:16678-16683 .
Free Online Full-text Article
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
,
|
