From the School of Biological Sciences, Life Sciences Building, University of Liverpool, P. O. Box 147, Liverpool L69 7ZB, United Kingdom
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ABSTRACT |
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The YOR163w open reading frame on chromosome XV
of the Saccharomyces cerevisiae genome encodes a member of
the MutT motif (nudix hydrolase) family of enzymes of
Mr 21,443. By cloning and expressing this gene
in Escherichia coli and S. cerevisiae, we have
shown the product to be a (di)adenosine polyphosphate hydrolase with a
previously undescribed substrate specificity. Diadenosine 5',5 Recent interest in the diadenosine polyphosphates
(ApnA)1 has focused
on their possible roles as regulators of cell proliferation. Ap3A has been proposed as a component of the
interferon-induced antiproliferative response in mammalian cells (1),
whereas Ap4A, the intracellular level of which has long
been known to be associated with proliferation (2, 3), may be an
antagonist of this pathway. A key factor in this response in humans is
the fragile histidine triad (FHIT) protein, an Ap3A
hydrolase that is absent or defective in many common cancers (4, 5).
Precisely how this protein and its substrate, Ap3A,
contribute to antiproliferation is not clear, but there is little doubt
that the regulation of the intracellular levels of specific
diadenosine polyphosphates is of great importance.
Eukaryotic Ap3A hydrolases exhibit an approximately 10-fold
preference for Ap3A over Ap4A as
substrates2 (6). They are
members of the histidine triad (HIT) family of proteins and possess the
catalytic sequence motif HXHXHX in which the central histidine residue forms a covalent enzyme-AMP reaction intermediate (4). Animals and higher plants also possess an
asymmetrically cleaving Ap4A hydrolase that prefers
Ap4A but is also active toward higher homologues, such as
Ap5A and Ap6A, but inactive toward
Ap3A (6). This enzyme belongs to the MutT motif (or
nucleoside diphosphate linked to x (nudix)) family of nucleotide
hydrolases (7, 8). Together, these two enzymes are probably crucial for
regulating the Ap3A/Ap4A ratio.
Many lower eukaryotes appear unusual in possessing one or more
Ap4A phosphorylases in place of Ap4A hydrolase.
For example, Saccharomyces cerevisiae has two
Ap4A phosphorylases, Apa1 and Apa2, in addition to an
Ap3A hydrolase (6, 9-11). Like the Ap4A
hydrolases, the yeast phosphorylases can also degrade Ap5A but not Ap3A (6, 11). The phosphorylases appear to be
distantly related to the HIT proteins, having an
HXHXQ motif in place of the
HXHXH histidine triad (10, 12). Although S. cerevisiae does not have an Ap4A hydrolase, genes for
five potential MutT motif proteins can be discerned in the genomic
sequence. Here, we report that one of these, YOR163w (GenBankTM
accession no. Z75071) from chromosome XV, encodes an Ap6A
hydrolase that is also active against Ap5A and the
adenosine 5'-polyphosphates p5A and p4A, but
not Ap4A or Ap3A. This is the first time that
an enzyme with this substrate specificity has been described. A
preliminary report of this work has appeared (13).
Materials
Ap6A was synthesized by carbodiimide condensation of
ATP (14). p5A was synthesized using the recombinant LysU
lysyl-tRNA synthetase and tetrapolyphosphate (15, 16). The plasmid
pXLys5 was a gift from P. Plateau, and the LysU protein was purified as
described (16). All other nucleotides were from Sigma. The cosmid clone
pUOA1258, which carries the complete YOR163w open reading frame from
yeast chromosome XV, was a gift from B. Dujon. The vector pPGY1 was a
gift from L. D. Barnes. Calf intestinal alkaline phosphatase (2000 units/mg) and yeast inorganic pyrophosphatase (200 units/mg) were from
Boehringer Mannheim. Pfu DNA polymerase was from Stratagene.
H218O (97.66 atom %) was from Amersham
Pharmacia Biotech.
Methods
Cloning in Escherichia coli--
The YOR163w gene was amplified
from the cosmid clone pUOA1258 (GenBankTM accession no.
U55021) using the polymerase chain reaction. The 29-mer oligonucleotide
primers d(AACACACCATGGGCAAAACCGCGGATAAT) and
d(AGGAATGGATCCATATGTTTGCGGTGGCT) were synthesized to provide an
NcoI restriction site at the start of the amplified gene and a BamHI site at the end. After amplification with
Pfu DNA polymerase, the DNA was recovered by
phenol/chloroform extraction and digested with NcoI and
BamHI, and the gel-purified restriction fragment was ligated
into the NcoI and BamHI sites of the pET15b
vector (Novagen), thus regenerating the ATG initiator in the
NcoI site and eliminating the His tag sequence from the
vector. The resulting plasmid, pET163W, was used to transform E. coli XL1-Blue cells for propagation.
Cloning in Yeast--
The YOR163w gene was amplified as
above using the primers d(GTGGGGGAATTCAAAATGGGCAAAACCGC) and
d(GAATAGCTCGAGATGTTTGC GGTGGCTTG). These primers provided an
EcoRI restriction site at the start of the amplified gene
and a XhoI site at the end. After amplification, the
recovered DNA was digested with EcoRI and XhoI,
and the gel-purified restriction fragment was ligated into the
EcoRI and XhoI sites of the yeast centromere
vector, pPGY1. The resulting construct, pPGY163W, generated the ATG
initiator downstream of GAL1p, a galactose-inducible promoter. The
plasmid was used to transform E. coli XL1-Blue cells for propagation.
Protein Expression in E. coli and Purification--
E.
coli strain BL21(DE3) was transformed with pET163W. A single
colony was picked from an LB agar plate containing 20 µg/ml ampicillin and inoculated into 10 ml of LB medium containing 60 µg/ml
ampicillin. After overnight growth, the cells were transferred to 1 liter of LB medium containing 60 µg/ml ampicillin and grown to an
A600 of 0.9. Isopropyl-1-thio- Protein Expression in Yeast and Purification--
S
cerevisiae strain INVScI was transformed with pPGY163W. A single
colony was picked from an SC-Ura (Synthetic Complete medium without
uracil) agar plate and inoculated into 100 ml of SC-Ura medium
supplemented with 5% glucose. After 36 h, the cells were harvested by centrifugation, resuspended in 1 liter of SC-Ura (5%
glucose), and further grown for 24 h. The cells (4.27 g) were again recovered by centrifugation, resuspended in 1 liter of SC-Ura (2% galactose, 1% raffinose), and grown for 16 h to fully induce expression of YOR163w. The induced cells (8.1 g) were harvested, washed, and resuspended in 8 ml of breakage buffer (50 mM
Tris acetate, pH 7.5, 0.3 M NaCl, 10 mM
2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 5 µM
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, and 1 mM benzamidine). An equal volume of 0.5-mm glass
beads was added, and the cells were disrupted by shaking at 1600 rpm
for 10 min at 4 °C in a Mikro-Dismembrator U (B. Braun Biotech). The homogenate was decanted, and a cytosolic extract was recovered by
centrifugation at 100,000 × g at 4 °C for 1 h.
Crude extract (13.5 ml) was applied at 0.5 ml/min to a 50 × 15-mm
column of Ni2+-nitrilotriacetic acid-agarose (Sigma)
equilibrated with 50 mM Tris acetate, pH 7.5, 0.3 M NaCl, and 10 mM 2-mercaptoethanol. After
eluting the unbound protein, a linear gradient of 0-30 mM histidine in equilibration buffer was applied at 1 ml/min. Fractions (4 ml) were assayed for Ap6A hydrolytic activity, pooled, and dialyzed against 10 mM sodium phosphate, pH 6.8, 0.01 mM CaCl2. The dialysate (20 ml) was applied at
1 ml/min to a 100 × 7.8-mm Bio-Gel HPHT column (Bio-Rad), and the
protein eluted with a linear gradient from 10 mM sodium
phosphate, pH 6.8, 0.01 mM CaCl2 to 350 mM sodium phosphate, pH 6.8, 0.01 mM
CaCl2. Homogeneous YOR163w gene product eluted at 180 mM sodium phosphate.
Enzyme Assays and Product Identification--
Potential
substrates were screened by measuring the Pi released by
co-incubation of substrate with YOR163w protein and either inorganic
pyrophosphatase or alkaline phosphatase. The standard assay (200 µl)
with phosphomonoester substrates was incubated for 30 min at 37 °C
and contained 50 mM Bis-Tris Propane buffer, pH 6.9, 5 mM MgCl2, 1 mM DTT, 0.35 mM substrate, 1 µg (1 milliunit) YOR163w protein and 0.5 µg (100 milliunit) inorganic pyrophosphatase. Assays with
phosphodiester substrates contained 1 µg (2 units) alkaline
phosphatase instead of the pyrophosphatase. The Pi released in each case was measured colorimetrically (17). Chromatographic fractions were screened for activity with 100 µM
Ap6A using a rapid luminometric assay supplemented with 1 mM DTT (18). The reaction products were identified by high
performance ion-exchange chromatography. Assay mixtures (100 µl)
containing 50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 1 mM DTT, 0.16 mM substrate
and 1 µg of YOR163w protein were incubated for up to 10 min at
37 °C and applied to a 1-ml Resource-Q column (Amersham Pharmacia
Biotech) at 2 ml/min in 35 mM
NH4HCO3, pH 9.6. The elution system comprised
Buffer A (H2O) and Buffer B (0.7 M
NH4HCO3, pH 9.6, with a gradient of 5-100%
Buffer B over 10 min. Peaks were identified with the aid of standards
and quantified by area integration.
Kinetic parameters for p5A and p4A were
calculated by measuring the initial rate of product formation by high
performance liquid chromatography as described above. Because some of
the products of Ap6A and Ap5A breakdown are
also substrates for the enzyme, parameters for the dinucleotides were
calculated from the initial rate of adenosine production upon
co-incubation of the substrates with YOR163w protein and alkaline
phosphatase. Assays containing 50 mM Bis-Tris Propane
buffer, pH 6.9, 5 mM MgCl2, 1 mM
DTT, substrate (various concentrations), 0.2 µg (0.2 milliunit) of
YOR163w protein, and 0.4 µg (0.8 unit) of alkaline phosphatase were
incubated for 5-10 min at 37 °C and then applied at 1 ml/min to a
250 × 4.6-mm Phenomenex Jupiter C18 column in 4 mM
potassium phosphate, pH 6.1, 8% (v/v) methanol. The adenosine peak was integrated.
Determination of Site of Nucleophilic Substitution by Mass
Spectrometry--
A reaction mixture containing 100 mM
Bis-Tris Propane buffer, pH 6.9, 10 mM MgCl2, 2 mM DTT, and 6 µg of YOR163w protein was prepared in a
final volume of 50 µl. After freezing at Immunoblotting--
Protein extracts were analyzed on a 90 × 50 × 0.75-mm 15% SDS-polyacrylamide gel. The gel was
equilibrated immediately after electrophoresis in transfer buffer (10 mM CAPS-NaOH, pH 11.0, 10% (v/v) methanol) for 10 min
before electrophoretic transfer of the separated polypeptides to a
nitrocellulose membrane at 150 mA and 4 °C for 2 h. The
membrane was blocked for 2 h at room temperature with
phosphate-buffered saline containing 3% fat-free powdered milk and
0.2% Tween-20 and then probed with a 1:5000 dilution of whole rabbit
anti-YOR163w antiserum (raised by standard procedures) followed by a
1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit
second antibody. After washing, the membrane was developed by enhanced
chemiluminescence using the Amersham Pharmacia Biotech ECL kit.
Other Methods--
Protein concentrations were measured by the
Coomassie Blue dye binding method (19), using a mixture containing
equal weights of bovine serum albumin, conalbumin, cytochrome
c, and myoglobin as a standard.
Cloning and Expression of the YOR163w Gene Product
Open reading frame YOR163w potentially encodes a 188-amino acid
protein with a molecular mass of 21,575 Da. The intronless gene was
polymerase chain reaction-amplified from the cosmid clone pUOA1258
using 29-base forward and reverse primers that included NcoI
and BamHI sites, respectively, and the polymerase chain
reaction product was inserted into the pET-15b expression vector.
E. coli strain BL21(DE3) was transfected with the
recombinant plasmid, and exponentially growing cells were induced for
up to 3 h with isopropyl-1-thio--P1,P6-hexaphosphate
is the preferred substrate, and hydrolysis in
H218O shows that ADP and adenosine
5'-tetraphosphate are produced by attack at P
and AMP
and adenosine 5'-pentaphosphate are produced by attack at
P
with a Km of 56 µM
and kcat of 0.4 s
1. Diadenosine
5',5
-P1,P5-pentaphosphate,
adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also
substrates, but not diadenosine
5',5
-P1,P4-tetraphosphate
or other dinucleotides, mononucleotides, nucleotide sugars, or
nucleotide alcohols. The enzyme, which was shown to be expressed in log
phase yeast cells by immunoblotting, displays optimal activity at pH
6.9, 50 °C, and 4-10 mM Mg2+ (or 200 µM Mn2+). It has an absolute requirement for
a reducing agent, such as dithiothreitol (1 mM), and is
inhibited by Ca2+ with an IC50 of 3.3 mM and F
(noncompetitively) with a
Ki of 80 µM. Its function may be to
eliminate potentially toxic dinucleoside polyphosphates during sporulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside was added to 0.4 mM, and the cells were induced for 4 h. The induced cells (5.1 g) were harvested, washed, and resuspended in 50 ml of
breakage buffer (50 mM Tris-HCl, pH 8.0, 2 mM
EDTA, 0.1 M NaCl). The cell suspension was sonicated, and
the inclusion bodies were recovered by centrifugation at 10,000 × g for 20 min. After washing by resuspension in breakage
buffer containing 2.5 M urea, the inclusion bodies were
dispersed in 6 ml of 6 M guanidinium-HCl, 10 mM
DTT, and the extract was centrifuged at 100,000 × g
for 1 h. The supernatant was applied in 1-ml aliquots to a Bio-Rad Hi-Pore RP-304 reversed phase column (250 × 4.6 mm), and the
protein was eluted with a nonlinear gradient from 0.15% (v/v)
trifluoroacetic acid to 0.1% (v/v) trifluoroacetic acid, 80% (v/v)
CH3CN. Homogeneous YOR163w gene product eluted at 50%
(v/v) CH3CN.
70 °C, 50 µl of
ice-cold 2.0 mM Ap6A was added, and the mixture
was immediately returned to
70 °C and then lyophilized. The
reaction was initiated by reconstitution in 100 µl of
H218O, and Ap6A was hydrolyzed
completely to yield the major reaction products by incubation at
37 °C for 20 min. Products were separated by high performance
anion-exchange chromatography on a 1-ml Resource-Q column as described
above. Peaks, monitored by their absorbance at 259 nm, were collected
manually and lyophilized, and the distribution of the 18O
between AMP, ADP, and p4A was determined by positive ion
electrospray mass spectrometry. Masses were also determined for the
products of a control assay reconstituted in
H216O. Samples for electrospray mass
spectrometry were reconstituted in 20 µl of 50% (v/v) acetonitrile,
0.1% (v/v) formic acid and injected at 10 µl/min in to a VG-Quattro
quadrupole mass spectrometer (Micromass U.K., Ltd.). Analysis was
performed at a source temperature of 60 °C, capillary voltage of 3.1 kV, and cone voltage of 45 V and with the skimmer offset to 5 V. A scan
time of 1 s was employed, and the mass ranges of m/z
330-365 Da, m/z 420-455 Da, m/z 570-630 Da
were scanned for AMP, ADP, and p4A, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside.
SDS-polyacrylamide gel electrophoresis of cell extracts revealed the
presence in both the soluble fraction (10-20%) and inclusion bodies
(80-90%) of a major band migrating with an apparent molecular mass of
24 kDa, which increased with induction time and so was presumed to be the required product (Fig. 1). Inclusion
bodies were then isolated after 4 h of induction and solubilized,
and the protein was purified in a single step to homogeneity by
reversed phase chromatography (Fig. 2).
The N-terminal sequence of the purified recombinant protein was
determined to be GKTADNHGPVRS by Edman degradation, and its mass was
measured as 21,443 Da by electrospray mass spectrometry. These
correspond exactly to the predicted sequence and mass (21,443.6 Da) for
the 187-amino acid polypeptide lacking the N-terminal methionine. These
data confirm the accuracy of the cloning procedure and the identity of
the protein.
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Fig. 1.
Expression of YOR163w gene in E. coli BL21(DE3). Cells were transformed with pET163W as
described under "Experimental Procedures" and induced with 0.4 mM isopropyl-1-thio- -D-galactopyranoside
for up to 3 h. Samples were taken at hourly intervals, boiled in
sample buffer, and analyzed by SDS-polyacrylamide gel electrophoresis
in a 15% gel. The gel was stained with Coomassie Blue. Lane
1, 0 h; lane 2, 1 h; lane 3,
2 h; lane 4, 3 h. Protein standards: bovine serum
albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate
dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa),
-lactalbumin (14.2 kDa).
The presumed YOR163w gene product is indicated with an
arrow.
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Fig. 2.
Purification of YOR163w gene product.
YOR163w gene product was purified in a single step from solubilized
inclusion bodies as described under "Experimental Procedures."
- - - - -, acetonitrile gradient. Inset, a sample of the
pooled peak was analyzed by SDS-polyacrylamide gel electrophoresis in a
15% gel. Lane 1, protein standards as in Fig. 1; lane
2, 1 µg of YOR163w gene product.
In order to confirm that the observed properties of the enzyme were not due to an alternative folding of the protein after reversed phase chromatography, the enzyme was also overexpressed in a soluble form in a yeast host system and purified conventionally by chromatography on a nickel affinity resin and hydroxyapatite. The enzyme binds tightly to the nickel column even though it was not expressed with a histidine tag. Both procedures yielded enzyme with very similar properties. The data presented here were obtained with the bacterially expressed protein.
Properties of the Protein
Substrates--
Almost all MutT motif proteins studied so far are
nucleotide pyrophosphatases that hydrolyze compounds containing an NDP
linked to another moiety, hence the alternative name of nudix hydrolase (8). A wide range of nucleotides was assayed to determine the substrate(s) of the YOR163w protein. Of these, only Ap6A,
p5A, Ap5A, and p4A yielded
significant activity (Table I). ATP was very slowly degraded to ADP + Pi, whereas no activity was
detectable with the following compounds in the presence of
Mg2+ or Mn2+ ions: Ap4A,
Ap3A, Ap2A, NAD+, NADH,
NADP+, NADPH, desamino-NAD+, FAD, coenzyme A,
(deoxy)nucleoside 5'-triphosphates (GTP, CTP, UTP, ITP, dATP, dGTP,
dCTP, and TTP), nucleoside 5'-diphosphates (ADP, GDP, CDP, and UDP),
nucleoside 5'-monophosphates (AMP, GMP, CMP, and UMP), nucleotide
sugars (ADP-ribose, IDP-ribose, ADP-glucose, GDP-glucose, GDP-mannose,
CDP-glucose, UDP-glucose, UDP-galactose, and
UDP-N-acetylgalactosamine), or nucleotide alcohols
(CDP-glycerol, CDP-choline, and CDP-ethanolamine). Diguanosine
polyphosphates were not tested as substrates. Table I shows the kinetic
constants obtained with the active substrates. Km
values were all within the range 30-70 µM and had
apparent catalytic constants (kcat) below 1 s1. According to the calculated catalytic efficiencies
(kcat/Km), Ap6A
is an 8-fold better substrate than Ap5A, the overall
preference being Ap6A > p5A
p4A > Ap5A. Kinetic parameters for
p5A were calculated using subsaturating substrate
concentrations only because substrate inhibition by this compound was
observed above 50 µM. Given the preference for
Ap6A, we propose that this protein should be described as a
diadenosine
5',5
-P1,P6-hexaphosphate
hydrolase.
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Reaction Requirements--
With 160 µM
Ap6A as substrate, the enzyme displayed optimal activity at
pH 6.9, 50 °C, and 4-10 mM Mg2+.
Mn2+ at 200 µM also sustained optimal
activity, but Ca2+ inhibited with an IC50 of
3.3 mM. F was also inhibitory
(noncompetitive), with a Ki of 80 µM.
In this respect, the yeast Ap6A hydrolase is similar to but less sensitive than the plant and animal Ap4A hydrolases,
which have Ki values for F
in the
ranges 2-3 and 20-30 µM, respectively (20). The enzyme had an absolute requirement for a reducing agent, such as DTT (optimal
at 1 mM).
Reaction Products--
The reaction products generated from each
of the four active substrates were determined after various incubation
times by ion-exchange high performance liquid chromatography (Table I and Fig. 3). From the kinetics of product
formation, the following overall conclusions were drawn.
Ap6A yielded mainly p4A + ADP (76%) but also
p5A + AMP (24%). AMP must be a primary breakdown product
because ADP is resistant to further hydrolysis, hence the enzyme
displays two alternative modes of attack on the Ap6A substrate. p5A, either alone or as a product of
Ap6A breakdown, generated almost exclusively
p4A + Pi. The example high performance liquid
chromatography profile in Fig. 3 shows only a small amount of
p5A; however, assays using shorter incubation times clearly showed the generation of equimolar amounts of p5A and AMP
before the p5A itself is degraded to p4A + Pi. ATP was also observed, most likely due to the breakdown
of the primary p4A product (see below). Similarly,
Ap5A yielded predominantly p4A + AMP (96%), but with a small amount of ATP + ADP (4%), whereas p4A
broke down to ATP + Pi. The preferential generation of
p4A from both Ap6A and Ap5A
suggests a reaction mechanism similar to the plant and animal
Ap4A hydrolases, which always generate ATP from
ApnA substrates (n 4). A binding pocket on
these enzymes accommodates a pppA moiety, with the fourth phosphorus
distal to the A being subject to nucleophilic attack by water (21, 22).
By analogy, the yeast Ap6A hydrolase appears to
preferentially accommodate a ppppA moiety in the substrate binding
site.
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Regarding the generation of alternative products, this could occur in
one of three ways. Using Ap6A as an example, these are (i)
exclusive attack of the nucleophile (presumed to be water) on
P, with elimination of the
P
O(P
) bond, yielding p4A + ADP, and elimination of the P
O(P
) bond,
yielding p5A + AMP; (ii) attack on P
and
elimination of the P
O(P
) bond, yielding
p4A + ADP, and attack on P
and elimination
of the P
O(P
) bond, yielding
p5A + AMP; (iii) attack on P
and elimination
of the P
O(P
) bond, yielding
p4A + ADP, and attack on P
and elimination
of the P
O(P
) bond, yielding
p5A + AMP. These possibilities can be distinguished by
carrying out the reaction in the presence of
H218O and following the fate of the
18O by mass spectrometry (23) (Fig.
4). When Ap6A was hydrolyzed in the presence of H216O, the AMP and ADP
products had masses of 348 and 428 Da, respectively (Fig.
5, A and C),
whereas hydrolysis in the presence of H218O
resulted in fully 18O-labeled AMP and ADP, with masses of
350 and 430 Da, respectively (Fig. 5, B and D).
In both cases, the p4A product was unlabeled, with a mass
of 588 Da (Fig. 5, E and F), whereas rapid
degradation of the p5A prevented an assessment of its
labeling pattern. Because only mechanism iii leads to labeling of both
AMP and ADP and lack of labeling of p4A (Fig. 4), this must
be the normal mode of attack and is, therefore, identical to that
previously established for the Artemia and lupin
Ap4A hydrolases (22-24).
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Presence in Yeast-- A rabbit polyclonal antibody was raised against the recombinant YOR163w protein in order to confirm that this protein is normally expressed in yeast. Fig. 6 shows that exponentially growing yeast cells express a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with YOR163w. The native protein also migrates with an anomalously high mass of 24 kDa. This phenomenon has also been observed with the 16.7-kDa human Ap4A hydrolase, which usually migrates as 19-21 kDa (7).
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DISCUSSION |
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The recovery of active YOR163w protein after reversed phase
chromatography and its high temperature optimum of 50 °C reflect the
high stability of the MutT motif proteins as a family. This purification system has also been found to yield pure and fully active
recombinant human Ap4A
hydrolase.3 Stability can
probably be attributed to the mixed -sheet structure shown to be at
the core of the E. coli MutT protein itself and other highly
stable proteins (25).
So far, Ap6A and Ap5A have only been described
in the secretory granules of certain specialized mammalian cells. It is
not known whether they are present in the cytosol of eukaryotes in general, including yeast. If so, and if they are synthesized by aminoacyl-tRNA synthetases in a reaction similar to the lower order
diadenosine polyphosphates (26, 27), then both p5A and p4A would be required as adenylate acceptors. Neither of
these compounds exists at detectable levels in vegetative yeast cells; however, they are both synthesized and excreted during the latter stages of sporulation following ascospore formation, reaching 1.5 and
2% of the concentration of ATP, respectively (28). They are not
produced by asporogenous a/a or /
strains placed in sporulation
medium, and so they have been proposed as signals marking the end of
sporulation (28). They may be synthesized by acetyl-CoA synthetase,
which is known to generate them in vitro (29). Their
presence in yeast cells suggests that Ap6A and
Ap5A might also be synthesized at low levels during
sporulation. Because Ap5A is a potent inhibitor (active in
the nanomolar range) of the essential enzyme adenylate kinase (30), one
function of the Ap6A hydrolase may be to eliminate these
potentially toxic dinucleotides during sporulation. In this context, it
is of interest to note that the region upstream of the YOR163w gene
contains a single copy of the stress response element 5'-AGGGG, which
is known to contribute to the response to nitrogen starvation (31). The
possibility that YOR163w expression is regulated by cellular stress
remains to be determined. Alternatively, the accumulation of
p5A and p4A during this period may reflect a
higher activity of the Ap6A hydrolase during vegetative
growth, its function being the removal of the substrates for
Ap6A and Ap5A synthesis. Such functions would
be in keeping with the "housecleaning" role proposed for the nudix
hydrolase family (8).
In generating AMP + p5A from Ap6A, the
reaction mechanism of the yeast Ap6A hydrolase is identical
to that previously determined for the production of AMP + ATP by the
Artemia and lupin Ap4A hydrolases, namely
nucleophilic attack of water on P and elimination of the
P
O(P
) bond (22-24). Interestingly, the
Artemia Ap4A hydrolase can be forced to switch
attack to P
when presented with substrates containing
nonscissile P
C phosphonate linkages, such as
diadenosine
5',5
-P1,P4-(P1,P2-monofluoromethylene-P3,P4-monofluoromethylene)
tetraphosphate (ApCHFppCHFpA) (21), or
-thiophosphates, such as
(Rp,Sp)-diadenosine
5',5
-P1,P4-(P1,P4-dithio)-tetraphosphate
(ApspppsA) (32). This so-called frameshift mode
of attack was attributed to a flexibility in the binding of the
polyphosphate substrate to the enzyme, with either P
or
P
being positioned next to a fixed catalytic center, a
situation more commonly encountered with polymeric substrates (21).
This flexibility is also demonstrated by the yeast Ap6A
hydrolase with the natural substrate Ap6A and, to a lesser
extent, Ap5A, with alternative sets of products being
generated in each case.
Fig. 7A shows a partial sequence alignment of the YOR163w protein with other known eukaryotic dinucleoside polyphosphate hydrolases, including the YA9E protein from Schizosaccharomyces pombe, which shares 43% sequence identity with YOR163w. The gene encoding YA9E has recently been cloned and expressed,4 and the protein has ApnA hydrolase activity with Ap6A and Ap5A as the preferred substrates, but with some activity toward Ap4A, in contrast to YOR163w, which has no activity with this substrate. Several observations can be made. First, YOR163w has an extra proline residue inserted in the MutT motif, the sequence common to all members of this protein family. This may in part explain the exclusive accommodation of the longer polyphosphate chains compared with the Ap4A hydrolases and the YA9E protein, which do not have this extra residue. Second, the hydrophobic patch in the fungal enzymes located just N-terminal to the MutT motif (YOR163w residues 47-50) is more similar to that in the animal Ap4A hydrolases than the plant enzyme sequences (Fig. 7A). However, further toward the N terminus (YOR163w residues 26-40), both of the fungal proteins, especially YOR163w, share additional sequence similarity with the two plant Ap4A hydrolases. This similarity is absent from the animal hydrolases, which do not align with any significance in this region. The fungal and plant enzymes also share the enzymic property of hydrolyzing both nucleoside and dinucleoside polyphosphates: the lupin Ap4A hydrolase degrades p4A, whereas this compound is a potent inhibitor of the animal Ap4A hydrolases (6, 33). Thus, there may be a closer evolutionary relationship between the plant and fungal ApnA hydrolases. Another activity that may be related is the dinucleoside polyphosphate hydrolase purified from the green alga Scenedesmus obliquus, an organism that, like S. cerevisiae, has an Ap4A phosphorylase (34). This enzyme hydrolyzes ApnA with the preference Ap5A > Ap4A > Ap6A. No significant similarity to the E. coli orf186 gene product, an enzyme that prefers Ap3A as substrate but that also hydrolyzes ADP-ribose and NADH (35), was detected outside the MutT motif.
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With regard to possible mammalian orthologs of YOR163w, a 41-amino acid sequence of this protein encompassing the MutT motif shows 50% identity and 65% similarity with two closely related but distinct sequences that are represented by several human, mouse, and rat clones in the GenBankTM expressed sequence tag data base. The alignment with the two human sequences is shown separately from the other dinucleoside polyphosphate hydrolases in Fig. 7B for clarity. One of these, DIPP, has recently been shown to be a diphosphoinositol polyphosphate phosphohydrolase, the first MutT motif protein with activity toward non-nucleotide substrates (36). Like YOR163w, DIPP shows positional flexibility in its site of attack. It is believed to attack a different pyrophosphoryl group in its two substrates, diphosphoinositol pentakisphosphate and bis(diphosphoinositol) tetrakisphosphate. Given the broad substrate specificity of some MutT motif proteins and the similarity between these proteins, it will be of particular interest to determine whether YOR163w can also hydrolyze diphosphoinositol polyphosphates.
In conclusion, we have characterized a dinucleoside polyphosphate nudix
hydrolase from S. cerevisiae with a novel substrate specificity. Given the current power of genetic manipulation in yeast,
phenotypic analysis of YOR163w knockouts in appropriate genetic
backgrounds (e.g. apa1 and apa2
mutants) should help determine the role(s) of this enzyme and the
relative contribution of hydrolases and phosphorylases to ApnA
catabolism and function in those organisms in which both types of
enzyme exist (34).
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ACKNOWLEDGEMENTS |
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We are grateful to M. C. Prescott for mass spectrometric determinations. We thank L. D. Barnes for communicating unpublished results and for valuable discussions and P. Plateau, B. Dujon, and L. D. Barnes for providing reagents.
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FOOTNOTES |
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* Financial support was provided by the Leverhulme Trust and the Royal Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 44-151-794-4369;
Fax: 44-151-794-4349; E-mail: agmclen{at}liv.ac.uk.
2 All the enzymes mentioned are active toward other dinucleoside polyphosphates in addition to diadenosine polyphosphates.
3 J. L. Cartwright and A.G. McLennan, unpublished observations.
4 S. W. Ingram, S. A. Stratemann, and L. D. Barnes, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
ApnA, diadenosine
5',5-P1,Pn-polyphosphate;
Ap2A, diadenosine
5',5
-P1,P2-diphosphate;
Ap3A, diadenosine
5',5
-P1,P3-triphosphate;
Ap4A, diadenosine
5',5
-P1,P4-tetraphosphate;
Ap5A, diadenosine
5',5
-P1,P5-pentaphosphate;
Ap6A, diadenosine
5',5
-P1,P6-hexaphosphate;
p5A, adenosine 5'-pentaphosphate;
p4A, adenosine 5'-tetraphosphate;
DTT, dithiothreitol;
nudix, nucleoside
diphosphate linked to x;
Bis-Tris propane, 1,3-bis[tris(hydroxyethyl)methylamino]propane;
CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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REFERENCES |
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