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INTRODUCTION |
Strains of the cyanobacterium Microcystis aeruginosa
sp. synthesize the cyclic heptapeptide microcystin-LR, a potent toxin toward humans and other animals (1, 2). In eukaryotes, the immediate
targets of microcystin-LR are the structurally homologous catalytic
subunits of the protein-serine/threonine phosphatases PP2A and
PP11 (3), to which it binds
with nanomolar or near nanomolar affinity, respectively (4).
Sensitivity to these and other toxins, such as okadaic acid, is so
highly conserved among eukaryotic PP1 and PP2A that it serves as a
criterion for the identification of these enzymes in cell extracts (5).
Moderate sensitivity to microcystin-LR extends to homologs recently
identified in members of the Archaea such as
Methanosarcina thermophila TM-1 (6, 7) and Pyrodictium abyssi TAG11 (8).
Recently, the presence of open reading frames potentially
encoding PP1/2A-like, also known as the PPP family (9), protein phosphatases has been reported in two strains of M. aeruginosa, the microcystin-producing strain M. aeruginosa PCC 7820 and the non-producing strain M. aeruginosa UTEX 2063 (10). These open reading frames have been
designated pp1-cyano1 and pp1-cyano2, respectively. Given the observation that microcystins accumulate within
the interior of toxin-producing cyanobacteria (11), and the near
absolute conservation of sensitivity to these compounds by members of
the PP1/2A superfamily (5), how do microcystin-producing cyanobacteria
protect themselves from the action of endogenous toxins against their
presumptive PP1/2A-like protein phosphatases? It has been suggested
that cyanobacteria produce these secondary metabolites to help ward off
encroachments by other microorganisms upon their habitat, thus gaining
a competitive advantage (12). Do the presumptive PP1/2A-like protein
phosphatases in strains of cyanobacteria that do not synthesize
microcystins differ in sensitivity from the equivalent enzymes in
toxin-producing strains? In order to answer these questions we
(a) obtained a complete clone of pp1-cyano1 and
determined the DNA-derived amino acid sequence of its predicted protein
product, PP1-cyano1; (b) expressed and purified PP1-cyano1
and PP1-cyano2 in order to study certain of their properties, including
their sensitivity to toxins; and (c) compared the properties
of the recombinant enzymes with those of PP1-cyano1 and PP1-cyano2
partially purified from their natural sources, M. aeruginosa
PCC 7820 and M. aeruginosa UTEX 2063.
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EXPERIMENTAL PROCEDURES |
Growth of Cyanobacteria--
M. aeruginosa PCC 7820 and M. aeruginosa UTEX 2063 were grown with continuous
aeration and lighting in BG-11 medium (13). When the late exponential
phase of growth was reached, the cells were harvested by centrifugation
and washed with Buffer A and stored at
20 °C.
Standard Procedures--
Protein concentrations were measured
according to the method of Bradford (14) using premixed reagent and a
standardized solution of bovine serum albumin from Pierce (Rockford,
IL). SDS-polyacrylamide gel electrophoresis was performed as described
by Laemmli (15). To visualize polypeptides, gels were stained with
Coomassie Brilliant Blue as described by Fairbanks et al.
(16).
Buffers--
Buffer A consists of 20 mM Tris, pH
8.0, containing 50 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. Buffer B
consists of 10 mM Tris, pH 8.0, containing 50 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/ml
leupeptin. Buffer C consists of 50 mM imidazole, pH 8.0, containing 100 mM NaCl, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 10% (v/v)
glycerol. Buffer D consists of 50 mM imidazole, pH 8.0, containing 3 mM MnCl2 and 1 mg/ml bovine serum
albumin. Buffer E consists of 20 mM Tris-HCl, pH 7.0, containing 500 mM NaCl and 100 mM imidazole.
Buffer F consists of 50 mM imidazole, pH 8.0, containing
100 mM NaCl and 10% (v/v) glycerol.
Preparation of Substrates for Assay of Phosphohydrolase
Activity--
[32P]Phosphoproteins were prepared as
described previously (17, 18). Partially phosphorylated
poly-L-histidine and poly-L-lysine were
prepared as described by Wong et al. (19). Low molecular weight phosphomonoesters such as p-nitrophenyl phosphate and
diadenosine tetraphosphate (Ap4A) were purchased from Sigma.
Assay of Protein Phosphatase Activity--
Phosphohydrolase
activity toward 32P-labeled phosphoproteins was determined
by a variation of the procedure described in Kennelly et al.
(17). Samples of PP1-cyano1 or PP1-cyano2 were incubated at 37 °C in
a final volume of 30 µl of Buffer D containing 2 µM substrate-bound [32P]phosphate. The reaction was
terminated, typically after a period of 15-90 min, by the addition of
100 µl of 20% (w/v) trichloroacetic acid, mixed, centrifuged for 3 min at 12,000 × g in a microcentrifuge, and 50 µl of
the supernatant liquid removed and counted for 32P
radioactivity in 1 ml of Scintisafe Plus 50% liquid scintillation fluid (Fisher Scientific, Pittsburgh, PA). Activity toward
[32P]phosphorylase a was determined according
to the procedure provided by the manufacturer (Life Technologies Inc.,
Gaithersburg, MD). Phosphatase activity toward partially phosphorylated
homopolymers of L-histidine and L-lysine were
performed essentially as described by Wong et al. (19) with
the assay buffer altered to Buffer D and the incubation temperature
increased to 37 °C.
Assay of Phosphohydrolase Activity toward Low Molecular Weight
Phosphomonoesters and Ap4A--
Phosphomonoesterase
activity toward p-nitrophenyl phosphate and other low
molecular weight phosphomonoesters was assayed essentially as described
by Howell et al. (18). However, the assay buffer was altered
to Buffer D and the incubation temperature was increased to 37 °C.
Pyrophosphatase activity toward Ap4A was measured by the
method of Plateau et al. (20), in which the adenine
mononucleotides formed by the pyrophosphorolysis of Ap4A
are hydrolyzed to adenine and organic phosphate using high levels of
alkaline phosphatase. Briefly, PP1-cyano1 or PP1-cyano2 was incubated
for varying times at a temperature of 37 °C in a volume of 50 µl
of Buffer D containing 1 mM Ap4A and 3 units of
alkaline phosphatase (U. S. Biochemical Corp., Cleveland, OH).
Reaction was terminated by the addition of 150 µl of DE52 cellulose
equilibrated in 50 mM imidazole, pH 8.0. After mixing and
centrifugation in a microcentrifuge at 12,000 × g for
3 min, 50 µl of the supernatant liquid was assayed for inorganic
phosphate using malachite green (21).
The site cleaved in Ap4A was determined by identifying the
adenine nucleotides formed following pyrophosphorolysis by the method
of Mangold (22). Briefly, samples of PP1-cyano1 or PP1-cyano2 were
incubated for varying times at a temperature of 37 °C in 50 µl of
Buffer D containing 1 mM Ap4A. Reaction was
terminated by adding 50 µl of 10 mM EDTA. A 10-µl
aliquot of the quenched assay mixture was then analyzed by thin layer
chromatography on a plate of LK2F microcrystalline cellulose (Whatman,
Clifton, NJ) that was developed with a mixture of 35:25:15:15:10
1-butanol, acetone, acetic acid, 5% (v/v) ammonium hydroxide, water.
Samples of Ap4A, ATP, ADP, and AMP were run in parallel as
standards. Adenine nucleotides were visualized by virtue of their
fluorescence when illuminated by UV light. Symmetric diadenosine
tetraphosphatases produce ADP as their sole hydrolysis product, while
asymmetric forms produce AMP and ATP.
Assay of Protein Phosphatase Activity following
SDS-PAGE--
Measurement of protein phosphatase activity following
SDS-PAGE was performed essentially as described by Burridge et
al. (23) using [32P]casein as substrate.
Partial Purification of PP1-cyano1 and PP1-cyano2 from M. aeruginosa--
The procedures for purifying PP1-cyano1 from M. aeruginosa PCC 7820 and PP1-cyano2 from M. aeruginosa
UTEX 2063 were identical. Fifty grams of frozen cells were thawed and
suspended in 5 volumes of Buffer A containing 1 µg/ml DNase I,
ruptured by sonication using six pulses, each of 1-min duration, of a
Sonifier model 185 sonic disrupter fitted with a large probe, then
centrifuged at 12,000 × g for 20 min at 4 °C. The
supernatant liquid was collected and passed through a column of
CM-Trisacryl. The flow-through was collected and loaded onto a 6.5 × 40-cm column of DE52 cellulose equilibrated in Buffer B. After
washing with Buffer B, the column was eluted with Buffer B containing
400 mM NaCl. The column eluate, DE-52 fraction I, was
dialyzed overnight against Buffer B, then applied to a 3 × 60-cm
column of DE52 cellulose equilibrated in Buffer B. The column was
washed with Buffer B, then developed with a linear gradient consisting
of 400 ml each of Buffer B and Buffer B containing 400 mM
NaCl. Fractions, 10 ml each, were collected and assayed for protein
concentration and protein phosphatase activity. Active fractions were
pooled, reduced in volume to 20 ml using a Centriprep-10 centrifugal
concentrator (Amicon, Beverly, MA), and applied, in 10-ml portions, to
a 5 × 100 cm column of Sephacryl S-200 that had been equilibrated
in Buffer C. The column was developed with Buffer C and fractions, 5 ml
each, collected and assayed for protein phosphatase activity. Fractions
exhibiting protein phosphatase activity were pooled, concentrated using
a Centriprep-10 centrifugal concentrator, and stored at
20 °C.
Cloning and Sequencing of pp1-cyano1--
Genomic DNA, 2 µg,
isolated from M. aeruginosa PCC 7820 as described previously
(10), was digested with HindIII according to the
manufacturer's protocols (Life Technologies Inc.). Restriction fragments were separated on a 0.8% (w/v) agarose gel, then transferred to a Magna nylon membrane (MSI, Boston, MA). The membrane was probed
with a partial clone of PP1-cyano1 (10) that had been radiolabeled by
PCR amplification from M. aeruginosa PCC 7820 genomic DNA in
the presence [
-32P]dATP (24). A single species roughly
2.7 kilobase pairs in length hybridized with the probe. Starting with
20 µg of DNA, HindIII restriction fragments corresponding
in size to that identified above were extracted from an agarose gel,
and ligated into plasmid vector pZERO (Invitrogen, Portland, OR) that
had been cut previously with HindIII. Transformation of
Escherichia coli with the ligation mixture and
identification of positive clones by colony lifts followed standard
procedures (25). DNA inserts from positive clones were digested with
restriction enzyme XbaI following the manufacturer's
protocols (Life Technologies Inc.), subcloned, and positive subclones
completely sequenced on both strands using Sequenase version 2.0 (U. S. Biochemical Corp.) and oligonucleotide primers (Life
Technologies Inc.).
Expression of Recombinant PP1-cyano1 and PP1-cyano2 in E. coli--
The complete structural genes for PP1-cyano1 and PP1-cyano2
were amplified by PCR using Pfu DNA polymerase (Stratagene, La Jolla,
CA). The primers were designed to introduce annealing sequences for
BamHI and PstI restriction sites at each
end. A common reverse primer was used for each:
5'-CCAGCTGCAGTATTAATCAGATTATCAACTA-3'. The forward primers were
5'-CGATGGATCCGTATGTTGTTTAGAAAAATAG-3' for PP1-cyano1 and
5'-CGATGGATCCGTATGTTTTTTAGAAACATAG-3' for PP1-cyano2. The
resulting PCR products were ligated into the expression vector pRSET C
(Invitrogen, Portland, OR), transformed into competent E. coli DH5
(Life Technologies Inc.), and plasmid isolated
therefrom. The plasmid-encoded protein phosphatase genes were sequenced
to verify the fidelity of PCR amplification, etc. Competent E. coli BL21(DE3) pLyS (Promega, Madison, WI) were transformed with
the plasmids, grown until they reached an OD600 of
0.6-1.0, then expression of PP1-cyano1 or PP1-cyano2 induced by the
addition of isopropyl-1-thio-
-D-galactopyranoside to a
final concentration of 0.4 mM. Cells were grown in the
presence of isopropyl-1-thio-
-D-galactopyranoside
overnight at a temperature of 30 °C, harvested by centrifugation,
then resuspended in 50 ml of 20 mM Tris-HCl containing 0.5 M NaCl, 1 mM imidazole, 1 mM
phenylmethylsulfonyl fluoride, 2 mg/ml lysozyme, and 100 µg/ml DNase
I and placed on ice for 30 min. The cells were then lysed by sonic
disruption and the resulting lysate clarified by centrifugation at
17,000 × g for 30 min. The supernatant liquid was
directly applied to a 1.5 × 20-cm column of chelating Sepharose
Fast Flow (Pharmacia-LKB, Uppsala, Sweden) that had been charged with
ZnSO4 and equilibrated in Buffer E. The column was
extensively washed with Buffer E, and adhering proteins eluted with
Buffer E in which the imidazole concentration had been increased to 250 mM. The high imidazole eluate was collected and dialyzed
versus Buffer F. The dialyzed material was then concentrated
by centrifugal ultrafiltration (Centriprep 10) to a volume of 1 ml and
portions, 0.2 ml, applied to a 1 × 60-cm column of Sephacryl
S-200 that had been equilibrated in Buffer F. The column was developed
with Buffer F. Fractions, 0.5 ml each, were collected and assayed for protein phosphatase activity. Active fractions were pooled and stored
at
20 °C until needed.
Nucleotide Sequence Accession Number--
The nucleotide
sequence for PP1-cyano1 has been submitted to GenBank, which has
assigned it accession number U80886. The nucleotide sequence and
accession number of PP1-cyano2 can be found in Shi and Carmichael
(10).
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RESULTS |
Cloning of the Complete Gene for and DNA-derived Amino Acid
Sequence of PP1-cyano1--
Using the
113-base pair PCR fragment
originally isolated by Shi and Carmichael (10) as probe, Southern
analysis indicated that only a single gene potentially encoding a
PPP-like protein phosphatase was present in the genome of M. aeruginosa PCC 7820 (data not shown). A full-length clone of the
pp1-cyano1 gene was isolated from the genomic DNA of
M. aeruginosa PCC 7820 and sequenced using conventional
methods. The complete, DNA-derived amino acid sequence of the predicted
protein product, PP1-cyano1, is shown in Fig.
1. PP1-cyano1 is predicted to consist of
a polypeptide 264 amino acids in length with a molecular mass of 30,426 daltons and an isoelectric pH of 5.8. The nucleotide sequence of the
presumptive coding region of pp1-cyano1 is 98% identical to
that of pp1-cyano2 from M. aeruginosa UTEX 2063. The predicted protein products of the two open reading frames share
98% identity at the amino acid level.

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Fig. 1.
Comparison of the DNA-deduced amino acid
sequence of PP1-cyano1 with representative PPP family protein
phosphatases. Shown is the DNA-derived amino acid sequence of
PP1-cyano1 from M. aeruginosa PCC 7820 (PP1-cyano1) aligned
with the corresponding regions of PrpA from E. coli (PrpA,
Ref. 26), PP1-arch2 from M. thermophila TM-1 (PP1-arch2,
Ref. 7), PP1 sds21 from Saccharomyces pombe (PP1 sds21, Ref.
50), and PP2A from rabbit (PP2A, Ref. 51). Amino acid identities
between PP1-cyano1 and other protein phosphatases are boxed.
The regions containing the three highly conserved motifs characteristic
of PPP family protein phosphatases and related phosphohydrolases (3,
29, 33) are indicated by the designation Motif I, II, or
III immediately above. The area containing key residues of
the toxin-binding domain of mammalian PP1 (52) is underlined
with asterisks. Alignment was performed by eye, relying
heavily on previous analyses of the conserved features of eukaryotic
PPP family protein phosphatases (3).
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In contrast to their very high degree of sequence identity to each
other, the predicted gene products of pp1-cyano1 and
pp1-cyano2 exhibit relatively low, typically 17-19%,
identity to their next closest homologs among the PPP family of protein
phosphatases (Fig. 1), whose most prominent members are PP1 and PP2A
(9). This was to be expected in those cases where the predicted
products of these cyanobacterial genes were compared with their
counterparts from eukaryotes or the Archaea. However, the
lack of noticeably greater identity to the only other well
characterized PPPs from the Bacteria, namely PrpA and PrpB
from E. coli (26), or the bacteriophage encoded PP-
(27)
was somewhat surprising. We postulated that the lack of an obvious
"family resemblance" to other bacterial or bacterially associated
PPPs might reflect the extremely deep nature of the branching between
the cyanobacteria and other members of the bacterial phylogenetic
domain (28). A computer-generated phylogenetic tree of representative
PPP-like phosphohydrolases and related enzymes (Fig.
2) appears to confirm this supposition.
It grouped pp1-cyano1 and pp1-cyano2 together with the other bacterial phosphohydrolases, including the diadenosine tetraphosphatases (29), while the archaeal and eukaryotic PPPs each
clustered together in a manner consistent with current three domain
models for phylogeny (28). Significantly, within the bacterial cluster,
PP1-cyano1 and PP1-cyano2 were grouped with the other known protein
phosphatases from or associated with bacteria, PrpA and PrpB from
E. coli (26) and PP-
(30), on a branch separate from that
containing the bacterial diadenosine tetraphosphatases.

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Fig. 2.
Phylogenetic analysis of the relationships
between PP1-cyano1 and PP1-cyano2 and selected phosphohydrolases of the
PPP family. Using the sequence regions bordered by conserved
Motifs I and III (see Fig. 1), a phylogenetic tree was determined using
the MegAlign program of Lasergene from DNA* (Madison, WI). The
numbers on the horizontal line indicate relative
evolutionary distance. Abbreviations used included: ApaH E. coli, diadenosine tetraphosphatase from E. coli (53);
ApaH S. typhimurium, diadenosine tetraphosphatase from
S. typhimurium (GenBank accession number Q56018); ApaH
K. aerogenes, diadenosine tetraphosphatase from Klebsiella
aerogenes (54); ApaH H. influenzae, putative
diadenosine tetraphosphatase from H. influenzae (55);
PrpA Anabaena, potential protein phosphatase from
Anabaena sp. PCC 7120 (40); PP Lambda, protein
phosphatase from bacteriophage (30); PrpA E. coli,
protein phosphatase from E. coli; PrpB E. coli,
protein phosphatase from E. coli (26);
PP1-cyano1, protein serine/threonine phosphatase 1 from
M. aeruginosa PCC 7820 (this study); PP1-cyano2,
protein serine/threonine phosphatase 1 from M. aeruginosa
UTEX 2063 (10); PP1-cyano3 (sll1387), protein
serine/threonine phosphatase 1 from Synechocystis PCC 6803 (39); PP1 Rabbit, protein serine/threonine phosphatase 1 from rabbit (56); PP1 sds, protein serine/threonine
phosphatase 1 from yeast (50); PPQ Yeast, protein
serine/threonine phosphatase 1 from yeast (57); PP2A Rabbit,
protein serine/threonine phosphatase 2A from rabbit (51);
PP2B 1, protein serine/threonine phosphatase 2B 1 from
human; PPT Yeast, protein serine/threonine phosphatase T
from yeast (58); PP1-arch1, protein serine/threonine
phosphatase 1 from Sulfolobus solfataricus (43); PPP
P. abyssi, serine/threonine specific protein phosphatase from
P. abyssi (8); PP1-arch2, protein
serine/threonine phosphatase 1 from M. thermophila TM-1
(7).
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Expression of PP1-cyano1 and PP1-cyano2 in E. coli and Purification
to Homogeneity--
The presumed structural genes for
pp1-cyano1 and pp1-cyano2 were cloned into vector
pRSET-C for expression in E. coli and subsequent evaluation
of the catalytic capabilities of the resulting polypeptide products.
The pRSET-C vector introduced an amino-terminal His6
sequence into the recombinant proteins, which facilitated their
purification by metal-chelate affinity chromatography (31). Recombinant
PP1-cyano1 and PP1-cyano2, which henceforth will be referred to as
rPP1-cyano1 and rPP1-cyano2, respectively, were purified to
electrophoretic homogeneity (Fig. 3) as
described under "Experimental Procedures" by
Zn2+-affinity chromatography followed by gel filtration
chromatography on Sephacryl S-200. An "in gel" protein phosphatase
assay using [32P]phosphoseryl casein revealed that each
preparation contained a single source of protein phosphatase activity
whose apparent Mr corresponded to that
calculated for the recombinant gene product (data not shown). The
position of the band of activity also corresponded with that of the
single polypeptide species visible following staining with Coomassie
Blue.

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Fig. 3.
Analysis of purified rPP1-cyano1 and
rPP1-cyano2 by SDS-PAGE. The genes encoding rPP1-cyano1 and
rPP1-cyano2 were expressed in E. coli and the resulting
protein products purified by metal-chelate affinity chromatography and
gel filtration chromatography as described under "Experimental
Procedures." Portions, 1 µg, of each preparation were analyzed by
SDS-PAGE on a 12% (w/v) acrylamide gel. Proteins were visualized by
staining with Coomassie Blue. At right is shown the
migration positions of protein standards. Lane 1 contains
rPP1-cyano1. Lane 2 contains rPP1-cyano2.
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Properties of Recombinant PP1-cyano1 and
PP1-cyano2--
rPP1-cyano1 and rPP1-cyano2 displayed divalent metal
ion-dependent protein phosphatase activity toward
[32P]phosphoseryl casein (Table
I). Mn2+ produced by far and
away the greatest stimulation of protein phosphatase activity, while
Mg2+, Co2+, and Ni2+ proved weakly
stimulatory. Cd2+, Cu2+, Fe2+, and
Zn2+ substantially inhibited protein phosphatase activity
when added along with the activating metal Mn2+.
Half-maximal activation of rPP1-cyano1 occurred at a Mn2+
concentration of approximately 0.2 mM (data not shown).
rPP1-cyano1 was active toward phosphoseryl casein over a pH range that
spanned from 6.5 to 9.5 (Fig. 4). Within
this range, optimal activity was exhibited from pH 7.5 to 9.0. The
enzyme was essentially inactive at pH values less than or equal to 5 or
greater than or equal to 10.
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Table I
Activation of cyanobacterial protein phosphatases by divalent metal
ions
The activity of purified recombinant rPP1-cyano1 and rPP1-cyano2, 10 ng
of each, along with that of partially purified PP1-cyanol (50 ng) and
PP1-cyano2 (75 ng) from M. aeruginosa PCC 7820 and M. aeruginosa UTEX 2063, respectively, was assayed under standard
conditions using [32P]phosphoseryl casein with the exception
that, where indicated, the compounds listed were substituted for the
activating divalent metal ion, Mn2+. All compounds were present
at a final concentration of 3 mM. Iron was maintained in
the ferric state by the addition of ascorbic acid to a final
concentration of 3 mM. All results are reported as the
percentage of activity relative to that observed with the most
activator, Mn2+.
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Fig. 4.
Influence of pH on the catalytic efficiency
of rPP1-cyano1 and PP1-cyano1. rPP1-cyano1 ( ), 10 ng, and
PP1-cyano1 ( ), 50 ng, were assayed for protein phosphatase activity
toward [32P]phosphoseryl casein under standard conditions
with the exception that the buffer composition and pH were varied as
indicated. Shown is the relative protein phosphatase activity detected
as a function of pH. For rPP1-cyano1, 100% activity was equal to 0.2 pmol of 32Pi released per minute, while the
corresponding value for PP1-cyano1 was 0.4 pmol/min. Buffer salts used,
all at a final concentration of 50 mM included sodium
acetate, pH 5.0; imidazole, pH 5.0-8.0; Tris-HCl, pH 8.0-9.0; and
glycine, pH 9.0-11.0.
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Next, the effects of various compounds known to inhibit protein
phosphatases and other phosphomonoesterases, as well as metabolites known to exert allosteric effects in bacteria, on the catalytic activity of rPP1-cyano1 and rPP1-cyano2 were determined (Table II). The various metabolites, such as
adenine nucleotides, cyclic AMP, sugar phosphates, glutamine, and
-ketoglutarate, were without effect. The same proved to be the case
for tartrate, an inhibitor of many acid phosphatases, and tetramisole,
the classic inhibitor of alkaline phosphatase. The general PPP family
protein phosphatase inhibitors fluoride and pyrophosphate proved
inhibitory toward PP1-cyano1 and PP1-cyano2 when present at millimolar
concentrations, as did orthovanadate. The latter is a general
phosphohydrolase inhibitor that displays particularly high potency
toward protein-tyrosine phosphatases, which are sensitive to
submillimolar concentrations of this phosphate mimetic.
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Table II
Effects of potential inhibitors/activators on the catalytic activity
PP1-cyano1 and PP1-cyano2
The activity of purified recombinant rPP1-cyano1 and rPP1-cyano2, 10 ng
of each, along with that of partially purified PP1-cyanol (50 ng) and
PP1-cyano2 (75 ng) from M. aeruginosa PCC 7820 and M. aeruginosa UTEX 2063, respectively, was assayed under standard
conditions, using [32P]phosphoseryl casein as substrate, with
the exception that the compounds listed were present at the indicated
final concentrations. All results are reported as the percent of
activity measured in the absence of added compounds.
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Microcystin-LR, okadaic acid, and calyculin A, the potent inhibitors of
homologous protein phosphatases from eukaryotes, PP1, and PP2A (4, 5),
proved innocuous even when present at the micromolar concentrations
known to inhibit the mildly sensitive eukaryotic PPP, calcineurin
(PP-2B). Ap4A also proved somewhat inhibitory when present
at millimolar concentrations. This compound is the substrate for a set
of bacterial phosphohydrolases, the diadenosine tetraphosphatases
(ApaH), that exhibit significant homology to the PPP family of protein
phosphatases (3, 29).
A survey of a broad range of phosphoesters and phosphoramides revealed
that rPP-cyano1 and rPP1-cyano2 exhibit multifunctional capabilities
in vitro. As expected based on their homology with eukaryotic members of the PPP family of protein phosphatases such as
PP1 and PP2A, both cyanobacterial enzymes dephosphorylated phosphoseryl
and phosphothreonyl residues on a variety of protein substrates
including casein, RCM-lysozyme, and myelin basic protein. Both enzymes
also dephosphorylated phosphotyrosyl residues on these same three
proteins. However, the phosphoseryl residue on glycogen phosphorylase
a did not serve as a substrate. Somewhat surprisingly, the
phosphoramide residues on partially phosphorylated homopolymers of
phospho-polylysine and 3-phospho-polyhistidine were readily
dephosphorylated. Using rPP1-cyano1, it was observed that the
steady-state rate of turnover measured for these phosphoramide substrates exceeded that measured for the best protein phosphomonoester substrate, phosphoseryl casein, by roughly 5-10-fold (Table
III). However, it should be noted that
these rate measurements were taken at a single, arbitrary concentration
of each substrate. Because of the differences in the sensitivities of
the assay procedures used, the concentration of the phosphoramide
substrates exceeded that of the protein phosphomonoesters by 500-fold.
rPP1-cyano1 also hydrolyzed p-nitrophenyl phosphate at a
significant rate.
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Table III
Relative activity of PP1-cyanol toward macromolecular and low molecular
weight organophosphates
The listed phosphoprotein, phosphopolypeptide, and low molecular weight
organophosphate compounds were tested as substrates for purified,
recombinant PP1-cyano1 (rPP1-cyano1) and PP1-cyano1 partially purified
from M. aeruginosa PCC 7820 (PP1-cyano1). All assays were
carried out as described under "Experimental Procedures." For
protein and polymer substrates, the type of phosphoamino acid(s)
present is given in parentheses. Phosphoprotein substrates were assayed
at a final concentration of 2 µM protein-bound
[32P]phosphate. Partially phosphorylated polylysine and
polyhistidine, p-nitrophenyl phosphate, and Ap4A
were assayed at a final concentration of 1 mM. All assays
were performed in triplicate. The quantities of rPP1-cyano1 and
PP1-cyano1 used for the assay of activity toward phosphorylated forms
of casein, RCM-lysozyme, and myelin basic protein were 10 and 50 ng,
respectively. For assay of activity toward glycogen phosphorylase
a, polylysine, polyhistidine, and Ap4A the
quantities were increased to 20 and 100 ng while for pNPP
these levels were 40 and 200 ng. Activities are reported as nanomole of
phosphate released per minute per mg of protein plus or minus standard
error.
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Given the homology between PPP family protein phosphatases and
diadenosine tetraphosphatases (3, 29, 32-34), Ap4A was tested as a potential substrate for rPP1-cyano1 and rPP1-cyano2. Both
enzymes hydrolyzed Ap4A at rates comparable to
macromolecular substrates. Once again, it is important to note that the
concentration of Ap4A employed, 1 mM, far
exceeded that used or even achievable with radiolabeled phosphoprotein
substrates (2 µM). Analysis of the reaction products by
thin layer chromatography revealed that both cyanobacterial protein
phosphatases cleaved Ap4A in a symmetric manner to yield
two molecules of ADP (Table IV).
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Table IV
Analysis of adenine nucleotides produced when Ap4A is incubated
with cyanobacterial PPPs
Purified recombinant rPP1-cyano1 and rPP1-cyano2, 20 ng each, and
partially purified PP1-cyano1, (100 ng) and PP1-cyano2 (150 ng) from
M. aeruginosa PCC 7820 and M. aeruginosa UTEX
2063, respectively, were incubated with Ap4A and the hydrolysis
products analyzed by thin layer chromatography as described under
"Experimental Procedures." Listed below are the mobilities
(Rf), relative to the solvent front, of both
adenine di- and mononucleotide standards and the products formed
following incubation with the listed phosphohydrolase. Standards were
run in quadruplicate, and their mobilities are therefore reported plus
or minus standard error.
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Properties of PP1-cyano1 and PP1-cyano2 Isolated from Native
Organisms--
Bacterial expression of the catalytic subunit of
mammalian PP1 results in the production of a recombinant enzyme that
exhibits a number of aberrant functional properties. These include a
dependence on the presence of exogenous divalent metal ions for
catalytic activity (35, 36) as well as a marked elevation in activity toward phosphotyrosyl proteins (37). Restoration of the recombinant mammalian enzyme to its native conformation can be accomplished by
incubation with its regulatory subunit, inhibitor-2, which apparently
functions as a molecular chaperone (37, 38). In this study, however,
incubation with mammalian inhibitor-2 had no effect on the catalytic
activity, substrate specificity, or other functional properties of
rPP1-cyano1 or rPP1-cyano2 (Data not shown).
To definitively establish whether the multifunctional capabilities and
other functional properties exhibited by rPP1-cyano1 and rPP1-cyano2
accurately reflected the properties of the native enzymes, we partially
purified these enzymes from their natural sources, the cyanobacteria
M. aeruginosa PCC 7820 and M. aeruginosa UTEX
2063, respectively. The major protein-serine/threonine phosphatase activities from both of these cyanobacteria were partially purified by
a combination of ion-exchange and gel-filtration chromatography as
described under "Experimental Procedures." Fig.
5 displays the results obtained with
extracts from M. aeruginosa PCC 7820. Similar results, not
shown, were obtained with extracts from M. aeruginosa UTEX
2063. In each case, a major peak of phosphohydrolase activity was
observed that eluted at a position similar, allowing for the
His6-containing NH2-terminal sequence
introduced by the pRSET-C vector system, to that of rPP1-cyano1 and
rPP1-cyano2. Analysis of the peak fractions using an in gel protein
phosphatase assay employing [32P]phosphoseryl casein as
substrate revealed the presence of a single catalytic species in each
with an Mr matching that estimated for
PP1-cyano1 or PP1-cyano2 (data not shown). On each column, the
phosphohydrolase activities toward phosphoseryl casein, phosphotyrosyl casein, 3-phospho-polyhistidine, phospho-polylysine, and
Ap4A coeluted, indicating that the native forms of
PP1-cyano1 and PP1-cyano2 possess multifunctional potential (Fig.
5).

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Fig. 5.
Coelution of protein phosphomonoesterase,
phosphoramidase, and diadenosine tetraphosphatases activity during
ion-exchange and gel filtration chromatography. Top,
DE52 fraction I was isolated from M. aeruginosa PCC 7820 and
dialyzed as described under "Experimental Procedures." DE52
fraction I, 1 liter containing approximately 1 g of protein, was
applied to a 3 × 60-cm column of DE52 cellulose that was washed
and developed with a linear salt gradient of 50-400 mM
NaCl. Fractions, 10 ml, were collected and portions, 15 µl each,
assayed for phosphohydrolase activity toward the following substrates
using standard procedures: , phosphoseryl casein; ,
phosphotyrosyl casein; , phospho-polylysine; ,
3-phospho-polyhistidine; and , Ap4A. Shown is the
relative phosphohydrolase activity in each fraction in either
counts/min of [32P]phosphate released for phosphoseryl
casein and phosphotyrosyl casein, or the OD660 of the
phosphate complex detected using malachite green for all other
substrates. The relative protein concentration ( ) is reported as the
OD595 of the protein-Coomassie Blue complex formed in the
protein assay of Bradford (9). The shape of the salt gradient is
indicated by the conductivity of each fraction (+). Bottom,
a portion, 10 ml, containing 10 mg of protein, of the active
fractions from the DE52 column, described above, protein were applied
to a 5 × 100-cm column of Sephacryl S-200. The column was
equilibrated and developed as described under "Experimental
Procedures." Fractions, 5 ml, were collected and portions, 15 µl
each, assayed for phosphohydrolase activity toward the following
substrates using standard procedures: , phosphoseryl casein; ,
phosphotyrosyl casein; , phospho-polylysine; ,
3-phospho-polyhistidine; and , Ap4A. Shown is the
relative phosphohydrolase activity in each fraction as well as the
relative protein concentration ( ), as indicated for Fig. 5,
top.
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The estimated molecular mass of PP1-cyano1, as determined by comparing
its elution on a Sephacryl S-200 column with that of protein standards,
was 29 kDa. This indicates that the native enzyme, at least in its
catalytically active form, is a monomer. A detailed comparison of the
divalent metal ion dependence (Table I), inhibitory spectrum (Table
II), pH activity profile (Fig. 4), and substrate specificity (Table
III, Fig. 5) of the partially purified enzymes from M. aeruginosa PCC 7820 or M. aeruginosa UTEX 2063 with
that of their recombinant counterparts indicated that rPP1-cyano1 and
rPP1-cyano2 accurately embodied the properties of the native enzymes.
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DISCUSSION |
With the determination of the complete gene sequences for a number
of bacterial organisms, it has become apparent that open reading frames
potentially encoding homologs of the major family protein-serine/threonine phosphatases in eukaryotes, the PPP family, are widespread throughout the Bacteria (39, 40). At present, however, little is known concerning the physical or functional properties of these potential bacterial protein phosphatases. It was
particularly intriguing to observe that the cyanobacteria contain open
reading frames whose predicted products resembled the catalytic
subunits of PP1 and PP2A, since these organisms produce peptide toxins
such as microcystin-LR that potently inhibit these enzymes in
eukaryotes (1, 2). The characterization of PP1-cyano1 from the
microcystin-LR producing cyanobacterium M. aeruginosa PCC
7820 and of PP1-cyano2 from the nonproducing strain M. aeruginosa UTEX 2063 represents an important step in the
exploration of potential signal transduction enzymes in the Bacteria (41).
The nucleotide sequences of pp1-cyano1 and
pp1-cyano2, and the resulting DNA-derived amino acid
sequences of their polypeptide products, were found to be 98%
identical. Thus, it was not surprising that their functional properties
were quite similar as well. Characterization of recombinant forms of
the proteins produced in E. coli permitted us to work with
homogeneous preparations whose origins could be unambiguously traced to
a defined gene product. Parallel experiments using partially purified
preparations of the enzymes from their natural sources, M. aeruginosa PCC 7820 and M. aeruginosa UTEX 2063, respectively, ensured that the behaviors displayed by the recombinant
gene products faithfully reflected those exhibited by the native
proteins. This represented a very significant potential pitfall, as
early attempts to express mammalian PP1 in E. coli had led
to the production of a catalytically active protein product that
displayed a number of aberrant functional properties (35-38). In the
case of PP1-cyano1 and PP1-cyano2, however, the behavior of the
recombinantly produced proteins mirrored that of their naturally
produced counterparts, and no attempt will be made to differentiate
between the two forms in the remarks that follow.
PP1-cyano1 and PP1-cyano2 are divalent metal ion-dependent
phosphohydrolases. The most effective activator tested was
Mn2+. In this property PP1-cyano1 and PP1-cyano2 closely
resemble the bacterially associated PPP, PP-
from the bacteriophage
gt11 (30, 33, 42), and the PPP homologs recently characterized in
the archaeons: i.e. PP1-arch1 from Sulfolobus
solfataricus (17, 43), PP1-arch2 from M. thermophila
TM-1 (6, 7), and Py-PP1 from P. abyssi TAG11 (8). The
inhibitory spectrum of PP1-cyano1 and PP1-cyano2 appears to be fairly
typical for members of the PPP superfamily of protein phosphatases,
with the notable exception that both enzymes were resistant to potent
inhibitory toxins such as okadaic acid and microcystin-LR, even when
these compounds are present at very high levels. The toxin
insensitivity of PP1-cyano1 renders the microcystin-LR producing
cyanobacterium M. aeruginosa PCC 7820 immune to the effects
of this endogenous toxin, which accumulates at various locations,
including the thylakoid and nucleoid, inside the cell (11).
Interestingly, PP1-cyano1 from the non-toxin producing strain M. aeruginosa UTEX 2063 also proved insensitive to these compounds,
suggesting that if these secondary metabolites serve as a means for the
molecular defense of environmental habitat (12), they are not directed
against the encroachments of other cyanobacteria. The bacteriophage
encoded PP-
also is insensitive to these compounds (30). However,
toxin insensitivity is not a general property of prokaryotic (as
opposed to bacterial) PPPs, since two of the three archaeal PPP family protein phosphatases characterized to date, PP1-arch2 (6, 7) and Py-PP1
(8), are toxin inhibitable.
When challenged with a variety of phosphate-containing substrates,
PP1-cyano1 and PP1-cyano2 displayed a wide range of hydrolytic capabilities. They hydrolyzed phosphoseryl and phosphothreonyl bonds in
a variety of protein substrates, with the noteworthy exception of
glycogen phosphorylase a. PP1-cyano1 and PP1-cyano2 also
displayed significant activity toward macromolecular substrates containing phosphotyrosine, 3-phosphohistidine, and phospholysine. Interestingly, PP-
had previously been reported to possess both phosphotyrosine and phosphohistidine phosphatase activity (33), while
PrpA and PrpB from E. coli, two partially characterized PPPs
of bacterial origin, also exhibited protein-serine and protein-tyrosine phosphatase activity in vitro (26). PP1, PP2A, and PP2C from eukaryotes also have been reported to exhibit protein-histidine phosphatase in vitro (44). However, data establishing
physiological relevance for this activity remains lacking several years
after this observation was reported, and these enzymes are still
classified as phosphoserine- and phosphothreonine-specific.
The prevalence of histidine-phosphorylated proteins in bacteria
(45, 46), including the phosphohistidyl proteins of the two-component
regulatory system and phosphoenolpyruvate:sugar phosphotransferase
system, raises the possibility that the multifunctional potential of
PP1-cyano1, PP1-cyano2, and/or other bacterial members of the PPP
family protein phosphatases may be realized in bacterial organisms,
however. This supposition is reinforced by the observation that genetic
manipulations of the genes for PrpA and PrpB in E. coli
perturbed both phosphohistidine levels and two-component signaling
events in this bacterium (26). The heterogeneous distribution of the
various protein phosphatase archetypes, i.e. PPP, PPM, PTP,
and low MW PTP, among the prokaryotes also implies that a greater
degree of catalytic versatility is required of these enzymes than of
their highly specialized eukaryotic counterparts (39). It is tempting
to extrapolate that PP1-cyano1 and PP1-cyano2, along with other
bacterial PPPs, may act on the phosphoaspartyl residues present on the
response regulator modules of the two-component system. However,
biochemical data indicating phosphoaspartyl phosphatase activity is
lacking, and the identification of distinct sources of phosphoaspartyl
phosphatase activity has been reported in the literature (47-49).
The PPP family of phosphohydrolases is not limited to protein-specific
phosphomonoesterases. The PPP family also shares conserved sequence
features with the diadenosine tetraphosphatase family of bacterial
pyrophosphatases that act on Ap4A (3, 29, 32-34). Both
PP1-cyano1 and PP1-cyano2 hydrolyzed Ap4A at a significant rate in vitro relative to phosphomonoester and phosphoramide
substrates. Two lines of evidence suggest that this represents a latent
or vestigial activity of protein phosphatases rather than their primary natural function. First and foremost, Ap4A only weakly,
30-50%, inhibited the dephosphorylation of
[32P]phosphoseryl casein, despite the fact that
(a) it was present at a 500-fold higher concentration than
the protein-bound phosphoryl groups and (b) phosphoseryl
casein is a physiologically irrelevant substrate that is not found in
cyanobacteria. One would expect a dedicated diadenosine
tetraphosphatase to exhibit a strong preference for its natural
substrate over an exogenous mammalian phosphoprotein, particularly when
the natural substrate is present in gross excess. Second, sequence
comparisons group PP1-cyano1 and PP1-cyano2 with known protein
phosphatases such as PP-
, PrpA, and PrpB on a branch distinct from
that encompassing the bacterial adenosine tetraphosphatases (Fig.
2).
While perhaps physiologically irrelevant, the ability of PP1-cyano1 and
PP1-cyano2 to act as both phosphomonoesterases and pyrophosphatases
in vitro possesses significant evolutionary implications. Many investigators have proposed an ancestral link between the adenosine tetraphosphatases and the PPP family of protein phosphatases based on comparisons of primary sequence data (3, 29, 32-34). The
results of the assays reported in this study provide the first functional evidence supporting both the proposed ancestral linkage of
these enzymes and the conserved nature of the catalytic mechanism which
they employ for the hydrolysis of phosphomonoester or pyrophosphate bonds.