From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received for publication, August 18, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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Nucleotide
pyrophosphatases/phosphodiesterases (NPPs) generate nucleoside
5'-monophosphates from a variety of nucleotides and their derivatives.
Here we show by data base analysis that these enzymes are conserved
from eubacteria to higher eukaryotes. We also provide evidence for the
existence of two additional members of the mammalian family of
ecto-NPPs. Homology searches and alignment-assisted mutagenesis
revealed that the catalytic core of NPPs assumes a fold similar to that
of a superfamily of phospho-/sulfo-coordinating metalloenzymes
comprising alkaline phosphatases, phosphoglycerate mutases, and
arysulfatases. Mutation of mouse NPP1 in some of its predicted
metal-coordinating residues (D358N or H362Q) or in the catalytic
site threonine (T238S) resulted in an enzyme that could still form the
nucleotidylated catalytic intermediate but was hampered in the second
step of catalysis. We also obtained data indicating that the ability of
some mammalian NPPs to auto(de)phosphorylate is due to an intrinsic
phosphatase activity, whereby the enzyme phosphorylated on Thr-238
represents the covalent intermediate of the phosphatase reaction. The
results of site-directed mutagenesis suggested that the nucleotide
pyrophosphatase/phosphodiesterase and the phosphatase activities
of NPPs are mediated by a single catalytic site.
Mammalian nucleotide pyrophosphatases/phosphodiesterases
(NPPs)12
are type II transmembrane proteins with a small intracellular domain
(10-80 residues) and a large extracellular domain (~830 residues)
that also harbors the catalytic site (1, 2). Since their catalytic site
is extracellular, this family of proteins are denoted as ecto-NPPs.
However, members of this family can also be processed into soluble,
secreted forms by proteolysis at specific sites C-terminal to the
transmembrane domain (3-5). In vitro, NPPs release
nucleoside 5'-monophosphates from nucleotides and a variety of
nucleotide derivatives. For example, NPPs efficiently hydrolyze ATP
into AMP and PPi. This catalysis occurs via a covalent intermediate, i.e. a nucleotidylated threonine in the
catalytic site (6). Some NPPs are also able to autophosphorylate and autodephosphorylate this catalytic site threonine. The
autophosphorylation is associated with an inhibition of the nucleotide
pyrophosphatase/phosphodiesterase activity (6).
The three known members of the mammalian ecto-NPP family have a
relatively broad tissue distribution and have been implicated in a
variety of cellular processes. NPP1, previously known as PC-1, is
likely to be involved in soft tissue calcification and bone
mineralization through its ability to generate PPi, an
inhibitor of calcification and mineralization (8). However, NPP1 has also been shown to oppose insulin signaling, and this effect does not
require a functional catalytic site (9). The anti-insulin effects of
NPP1 may be the result of its direct interaction with the insulin
receptor (10). NPP2 or autotaxin was shown to stimulate cell motility
(11) and to augment the invasive and metastatic potential of
ras-transformed cells (12). NPP3, also known as gp130RB13-6 (13) and B10 (5), has been reported to promote
the differentiation and invasive properties of glial cells (13).
Although most of the biological effects of NPPs appear to be mediated
by their NPP activity, the physiological NPP substrates remain to be
identified. It is our long term goal to obtain information on the NPP
substrate(s) by expressing mutants that can still form the catalytic
intermediate but can no longer hydrolyze this intermediate. Characterization of this "trapped" covalent adduct should then provide data on the nature of the substrate(s). As a first step toward
this goal, we report here on mutations that specifically block the
second catalytic step of NPPs. These "trapping" mutants were
designed on the basis of results of a structure-prediction analysis,
which suggested that the catalytic domain of NPPs is structurally and
catalytically related to that of well characterized enzymes such as
alkaline phosphatase. Unexpectedly, these studies have also revealed
that a single catalytic mechanism accounts for both the
nucleotide pyrophosphatase/phosphodiesterase and the auto
(de)phosphorylation activities of NPPs.
Sequence Analysis--
The organization of the genes encoding
human NPP2, human NPP4, and mouse NPP5 was determined by comparing the
respective cDNA sequences with the genomic sequences in the
GenBankTM data base, using the BLAST-2 sequences program at NCBI (14).
Intron-exon boundaries were verified according to Horowitz and Krainer
(15). The sequence of NPP from Candida albicans was obtained
from the Stanford Sequencing and Technology Center (available via the
World Wide Web). The sequences of NPPs from Caulobacter
crescentus and Porphyromonas gingivalis were acquired
from the Institute for Genomic Research (available via the World Wide
Web). The sequence of the Clostridium acetobutylicum NPP was
obtained from Genomic Therapeutics Corp. (available via the World Wide Web).
Generation and Expression of NPP Mutants--
Various mutants of
mouse NPP1 were expressed as HA-tagged fusion proteins in COS-1 cells
(Figs. 6 and 8). The expression vectors were generated by a two-step
procedure. First, the pSVL-SV40 vector was cut with XhoI and
BamHI, and ligated with an adaptor encompassing the sequence
of an HA tag (YPYDVPDYA) and a multiple cloning site, yielding the
pMB001 vector. The mouse NPP1 coding sequence, obtained from the
pSVL/PC-1 plasmid (17), was cut with XbaI and
BamHI and subcloned in the pMB001 vector. Point mutations
were introduced using the pMB001 vector and the QuickChange
site-directed mutagenesis protocol of Stratagene. PCR amplifications
were performed using Pwo proofreading polymerase (Roche
Diagnostics). All mutations were verified by DNA sequencing.
COS-1 cells were grown in Dulbecco's modified Eagle's medium
containing 10% (v/v) fetal bovine serum and 100 units/ml each of
penicillin and streptomycin. The cells were transfected with the NPP
constructs at 30-40% confluence, using the FuGeneTM6 reagent (Roche
Diagnostics). After 48-72 h the cells were washed twice in ice-cold
phosphate-buffered saline and lysed in 20 mM Tris/HCl at pH
7.5, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM benzamidine, 0.3 M NaCl, and 0.2% Triton
X-100. After centrifugation (5 min at 5000 × g), the
supernatant was used for immunoprecipitation of the HA-NPP fusions with
monoclonal HA tag antibodies (clone 12CA5) and protein A-TSK
(Affiland). The immunoprecipitates were washed once with 0.25 M LiCl and twice with Tris-buffered saline, resuspended in
50 mM HEPES at pH 7.5, and assayed for phosphodiesterase activity and for the ability to trap the covalent intermediates of the
phosphatase and the phosphodiesterase reactions.
Western Analysis--
Following Tricine-SDS-PAGE (7.5%) the
proteins were transferred onto polyvinylidene difluoride membranes
(Bio-Rad) by electroblotting at 38 V in 50 mM Tris-base
plus 50 mM boric acid at pH 8.3. Unspecific binding sites
were blocked in phosphate-buffered saline containing 5% (w/v) milk
powder and 0.2% (v/v) Triton X-100. Affinity-purified polyclonal
antibodies against a synthetic peptide encompassing the 14 C-terminal
residues of mouse NPP1 (LRLKTHLPIFSQED) were used to visualize the
HA-NPP fusion proteins. Peroxidase-labeled secondary antibodies were
used for chemiluminescent detection.
Phosphodiesterase Activity and Covalent Intermediates--
The
nucleotide phosphodiesterase activity was measured with
p-nitrophenyl thymidine 5'-monophosphate as substrate (6). The nucleotidylated intermediate, formed during the hydrolysis of 50 µM [ Preparation of [ A Family of NPP Proteins--
A search of the nonredundant
sequence data base at NCBI with NPP1-3, using the BLAST-algorithm
(21), resulted in the identification of two novel mammalian NPP
homologues, designated NPP4 and NPP5 (Fig.
1). An EST analysis revealed a rather
broad tissue distribution of these novel putative NPPs (data not
shown). Human NPP4 (453 residues) and NPP5 (477 residues) are
considerably smaller than NPP1-3 (863-925 residues), which can be
accounted for by the absence in NPP4-5 of N-terminal sequences
corresponding to the cytoplasmic tail, the transmembrane region, and
somatomedin-B-like domains of NPP1-3. In addition, NPP4 and NPP5 are
about 250 residues shorter at the C terminus. NPP4 and 5 are more
closely related to each other (51% identity) than to the corresponding
domain of NPP1-3 (29-37% identity). The genes encoding human NPP4
and NPP5 (GenBankTM accession no. AL035701) only contain 2 introns, as
compared with 24 introns for the NPP1-3-encoding genes (23, 24).
However, the two introns in the NPP4 and NPP5 genes coincide with
introns 13 and 15 in the genes encoding NPP1-3, providing additional
evidence for their common ancestry.
In addition to two novel mammalian NPPs, homology searches revealed the
existence of proteins ranging from bacteria to plants that shared a
significant similarity with the catalytic domain of the mammalian NPPs
(Fig. 2). The conservation included the catalytic site threonine and its surrounding residues (see consensus sequence in Fig. 1).
NPPs Belong to a Superfamily of Phospho-/Sulfo-coordinating
Metalloenzymes--
To identify more distant NPP relatives, the
nonredundant protein sequence data base at NCBI was searched with the
PSI-BLAST algorithm (27), using the sequence of the catalytic domain of mouse NPP1 (residues 192-568) as a query. Convergence was reached after 14 iterations, yielding proteins that could be clustered into
mainly five different enzyme families. Besides the NPP family, these
included the families of arylsulfatases (AS), phosphopentomutases, 2,3-bisphosphoglycerate-independent phosphoglycerate mutases (iPGM), and the alkaline phosphatases (AP). The latter four families have previously also been classified in a superfamily of
phospho-/sulfo-carbohydrate processing metalloenzymes, based on the
conservation of metal binding motifs (28). Furthermore, the crystal
structures of E. coli AP (PDB code 1ALK), human AS-A and
AS-B (PDB codes 1AUK and 1FSU, respectively) and Bacillus
stearothermophilus iPGM (PDB code 1EJJ) have been solved (29-32),
which has provided extensive proof of structural and catalytic similarities.
A PSI-BLAST search with the sequence of E. coli AP
(SwissProt P00634) attributed a higher similarity score to NPPs than to
AS (data not shown). Conversely, when the human AS-A sequence (SwissProt P15289) was used as query, NPPs emerged with a higher
similarity score than did alkaline phosphatases. Thus, within the
superfamily of phospho-/sulfo-coordinating metalloenzymes, the NPP
family is positioned intermediate between ASs and APs. Since the
catalytic domains of APs, ASs, and iPGMs have been shown to assume a
basically identical fold, i.e. a
Since a combination of various, independent prediction methods has been
shown to improve the reliability of secondary structure prediction (34,
35), a consensus secondary structure was predicted for the catalytic
domain of mouse NPP1, with the use of the PSI-PRED, PHD, SamT99, JPRED,
and Prof prediction algorithms (36-40). The predicted secondary
structure was aligned with the known secondary structure common to the
catalytic domains of APs, iPGMs, and ASs. This alignment revealed a
remarkable conservation in the length and position of The Reaction Mechanism of NPPs--
Since we predicted the
catalytic site of NPPs to have an arrangement similar to that of APs
and iPGMs, this also suggested a similar catalytic mechanism.
Transposing the known reaction mechanism of APs (44) and iPGMs (32) to
NPPs yields the reaction scheme shown in Fig. 5. It is proposed that
the Me2-activated nucleophilic hydroxyl of Thr-238 attacks the
phosphate of the incoming substrate, resulting in the generation of a
covalent, nucleotidylated intermediate. In the second catalytic step, a Me1-activated water molecule attacks this E-NMP intermediate, regenerating Thr-238 and releasing a nucleoside 5'-monophosphate. An
essential difference between NPPs and APs lies in the nature of their
respective substrates, i.e. a phosphodiester and a
phosphomonoester, respectively. In APs the substrate affinity is
increased by an arginine (Arg-166 in E. coli AP),
which is hydrogen-bonded to the two oxygen atoms of the phosphate
moiety that are not interacting with metal ions. In the typical NPP
substrates, however, one of these oxygens is bonded with the
nucleosidyl group (see Fig. 5). We suggest that binding of the latter
in a nucleosidyl binding pocket serves a function similar to that of
the arginine-mediated hydrogen bonding in APs.
Several lines of evidence provide support for the proposed reaction
scheme of NPPs. First, it has already been established that the NPP
reaction occurs in two steps and that a nucleotidylated intermediate is
formed with the catalytic site Thr (6). Second, it is well known that
NPPs are metalloenzymes since their activity is blocked by metal
chelators such as EDTA (47, 48). Third, the activity of NPP1 is blocked
by imidazole (18, 43) and by the histidine-acylator
diethylpyrocarbonate (results not shown), in keeping with the proposed
role of histidines in coordinating Me1 and Me2 (Figs.
4 and 5).
We have also obtained evidence in favor of the proposed reaction
mechanism by site-directed mutagenesis of NPP1. For that purpose mouse
NPP1 point mutants were expressed in COS-1 cells as HA-tagged fusion
proteins, immunoprecipitated from the cell lysates with anti-HA
antibodies, and assayed for NPP activity, using
p-nitrophenyl thymidine 5'-monophosphate as substrate. In
accordance with previous reports (9, 49, 50), mutation of the catalytic
site residue, i.e. T238S or T238A, decreased the NPP
activity by 95 and 100%, respectively (Fig.
6B). By contrast, mutation of
the neighboring residues (K237A and F239A) merely halved the activity.
Mutation of any of the 6 residues (Figs. 4 and 5) predicted to be
involved in the binding of Me1 (Asp-358, His-362, and His-517) or Me2
(Asp-200, Asp-405, and His-406) essentially abolished the NPP activity
(Fig. 6B). Mutation of the corresponding residues in
E. coli AP and B. stearothermophilus iPGM has
been reported to be inhibitory as well (41, 51-57).
It can be envisaged that the activity of NPP1 that is mutated in its
metal-coordinating residues, can be (partially) restored by the
addition of an excess of metals. Since the identity of the metals in
the catalytic site of NPPs is not known, we have first explored which
metals can restore the activity of NPP1 following metal chelation by 1 mM EDTA. We found that the activity of EDTA-inhibited NPP1
could be largely restored by the mere addition of 2-5 mM amounts of either ZnCl2 or CaCl2, but only
partially by 2-5 mM MgCl2 (data not shown). In
Fig. 6B it is shown that the activity of the H362Q and H517Q
mutants of NPP1 was also restored to about 60% of the control value by
the addition of 2 mM ZnCl2. The activity of the
other putative metal coordination mutants (D405N, H406Q, D358Q, D200N)
was also increased severalfold by the addition of Zn2+,
albeit to a lesser extent than that of the H362Q and H517Q mutants. As
expected, the activity of NPP1 mutated in the catalytic site residue
(T238A, T238S) or its flanking residues (K237A, F239A) were not at all
or only marginally affected by the addition of Zn2+. These
data provide additional proof for the role of Asp-200, Asp-358,
His-362, Asp-405, His-406, and His-517 in the coordination of metals
and also show that the deleterious effect of these mutations is not the
result of gross conformational changes.
We have also investigated whether the mutants of NPP1 are blocked in
the first and/or second step of catalysis. For that purpose, we
compared their ability to trap the nucleotidylated catalytic intermediate, using [ NPPs Also Exhibit a Phosphatase Activity--
Having concluded
that NPPs have a catalytic site that is similar to that of APs, we
wondered whether the capability of NPPs to autophosphorylate and
autodephosphorylate (see Introduction) could perhaps reflect an
intrinsic phosphatase activity, in which autophosphorylated NPP
represents the covalent phospho-intermediate. We have obtained initial
data that support this view. First, autophosphorylation of NPP1 was
also observed with [
We have also explored the effects of point mutations at or near the
catalytic site on the ability of NPP1 to become autophosphorylated (Fig. 8). Mutations D358N and H362Q still
allowed a weak labeling, whereas mutation of any of the other residues
proposed to be involved in the binding of Me1 (His-517) and Me2
(Asp-200, Asp-405, and His-406) completely abolished the
autophosphorylation in the presence of [ Conclusions and Perspectives--
We have made use of the wealth
of information in protein data bases to gain some initial insight into
the catalytic mechanism of NPPs. Rather unexpectedly, this analysis has
revealed that NPPs belong to the superfamily of
phospho-/sulfo-coordinating metalloenzymes. The detailed knowledge of
the structure and catalytic mechanism of some of these enzymes has
helped us to identify residues in NPPs that are required for catalysis.
The essential role of these residues has subsequently been confirmed by
site-directed mutagenesis. Interestingly, mutation of some of the
residues that were predicted to be involved in the second catalytic
step (Thr-238, Asp-358, His-362) generated enzymes that accumulated the
nucleotidylated intermediate.
Our studies have also provided an entirely novel insight into the dual
function of NPPs, i.e. as nucleotide
pyrophosphatases/phosphodiesterases and as auto(de)phosphorylating
enzymes. We suggest that the ability of some NPPs to
auto(de)dephosphorylate is actually a reflection of an intrinsic
phosphatase activity, whereby the autophosphorylated enzyme represents
the covalent intermediate of the phosphatase reaction. Based upon the
relative production rates of ADP, AMP, PPi, and
Pi by NPP1 (16) or NPP2 (58), it can be deduced that their
phosphatase and nucleotide pyrophosphatase/phosphodiesterase activities
are of similar order of magnitude. If anything, the contribution of the
phosphatase activity was underestimated in these studies since ADP, a
product of the phosphatase reaction, is also a substrate of the NPP
reaction. It should also be noted that the ratio of both enzymic
activities is bound to be substrate-dependent. Thus we
noted that p-nitrophenylphosphate, a classical substrate of
alkaline phosphatases, is not hydrolyzed to a measurable extent by NPP1
(data not shown).
The similarities between the catalytic core of NPPs and APs (Fig. 3),
combined with the results of site-directed mutagenesis (Fig. 8),
strongly suggest that the phosphatase and nucleotide pyrophosphatase/phosphodiesterase reactions are catalyzed by the same
catalytic site. At first glance, phosphatases and phosphodiesterases catalyze a completely different reaction. However, it can be argued that phosphatases hydrolyze phosphate esters whereas NPPs hydrolyze nucleotidyl esters (or acid anhydrides), resulting in the removal of
phosphate or nucleotidyl groups, respectively. Replacing a free
hydroxyl group of the substrate-bound phosphate moiety by a nucleoside
transforms a phosphatase reaction scheme into a phosphodiesterase reaction scheme. For NPPs to act both as nucleotide
pyrophosphatases/phosphodiesterases and as phosphatases, one only has
to imply that (some) substrates can be bound in two different ways. For
example, with ADP as substrate it is either the
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP by mouse NPP1 enzyme, was
trapped according to Blytt et al. (18), with slight
modifications (6). The trapped intermediate was visualized by
autoradiography, following 7.5% Tricine-SDS-PAGE. The
"autophosphorylation" of mouse NPP1 and of bacterial or bovine intestinal alkaline phosphatase (Roche Diagnostics) was followed during
an incubation at 30 °C in 100 mM HEPES at pH 7.5 and 50 µM [
-32P]ATP,
[
-32P]ADP, or [32P]Pi (Figs.
7 and 8). 5'-Nucleotidase from Crotalus atrox (3 units/ml; Sigma) was added to remove inhibitory AMP (16). The
"autophosphorylated" NPP or AP were visualized by autoradiography,
following 7.5% Tricine-SDS-PAGE.
-32P]ADP--
A mixture
containing 1.5 mM AMP, 1.5 mM
[
-32P]ATP, 5 mM HEPES at pH 7.5, 30 mM MgCl2, and 20 units/ml rabbit muscle
myokinase (Sigma) was incubated during 4 h at 37 °C.
Subsequently, the myokinase was denatured by heating in boiling water
for 3 min. Residual ATP and AMP were removed by an additional
incubation for 3 h at 37 °C in the presence of 10 mM glucose, yeast hexokinase (15 units/ml; Roche
Diagnostics), and 5'-nucleotidase from C. atrox (10 units/ml; Sigma). Finally, the [
-32P]ADP was purified
by the DEAE-chromatography procedure described by Tan (20).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
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Fig. 1.
Alignment of the catalytic domain of human
NPPs. The protein sequences were aligned using CLUSTAL X (22) and
the alignments were subsequently refined manually. The GenBankTM
accession numbers of the listed proteins are D12485
(HsNPP1), D45421 (HsNPP2), AF005632
(HsNPP3), AB020686 (HsNPP4), and AL035701
(HsNPP5). The latter represents a genomic sequence, but the
exonic sequences were derived by sequencing of a commercial mouse
cDNA clone (IMAGE clone 553842). The numbers indicate
the distance to the beginning and the end of each protein. Conserved
and identical residues are boxed. The figure also shows an
NPP "consensus sequence," as derived from an alignment of all
prokaryotic and eukaryotic NPPs that were obtained by data base
searching (see Fig. 2). In the consensus sequence, the following
symbols are used: , charged residue (D, E, K, R, N, Q);
, bulky
hydrophobic residue (I, L, V, M, F, Y);
, small residue (G, A, S,
T);
, acidic residues (D, E); ·, no particular residue. The
catalytic site threonine is underlined in the consensus
sequence.
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Fig. 2.
Phylogenetic conservation of NPPs. A
multiple alignment of the catalytic domain of the indicated NPPs was
generated by CLUSTAL X (22). Subsequently a phylogenetic tree was
generated using the neighbor-joining method (25) and was depicted using
version 1.6.1 of the Treeview program (26). The GenBankTM accession
numbers for the enlisted cDNAs or protein sequences are as follows:
HsNPP1, Homo sapiens NPP1 (D12485);
MmNPP1, Mus musculus NPP1 (J02700);
HsNPP2 (D45421); RnNPP2, Rattus
norvegicus NPP2 (D28560); HsNPP3 (AF005632);
RnNPP3 (Z47987); HsNPP4 (AB020686);
HsNPP5 (CAB56566); MmNPP5 (AF233377);
At1NPP, Arabidopsis thaliana 1 NPP (CAB45328);
At2NPP (CAB45329); At3NPP (CAB45330);
Ce1NPP, Caenorhabditis elegans 1 NPP (CAB02784);
Ce2NPP (CAB02785); Ce3NPP (AAC47919);
FpNPP, fowlpox virus (CAA07014); OsNPP,
Oryza sativa NPP (U25430); SpNPP,
Schizosaccharomyces pombe NPP (CAA22177); Sc1NPP,
Saccharomyces cerevisiae 1 NPP (AAB64493); Sc2NPP
(P25353); ZmNPP, Zymomonas mobilis NPP
(AAC70363). The sequence of CaNPP (C. albicans
NPP), CcNPP (C. crescentus NPP),
ClaNPP (C. acetobutylicum NPP), and
PgNPP (P. gingivalis NPP) were derived from
preliminary sequence data, as acknowledged under "Materials and
Methods."
-sheet sandwiched between
-helices (29-32), it seems likely that the catalytic domain of NPPs would adopt the same fold. In agreement with this view, the
fold recognition program GenTHREADER (33), which uses a threading
potential to evaluate the quality of fold assignments, proposes with
the highest level of confidence that the fold of the human AS-A and
AS-B occurs in the catalytic domain of human NPP1.
-helices and
-strands between these four families (Fig.
3A). By threading the sequence
of the predicted secondary structure elements of NPP1 onto the known
secondary structure of the AP, iPGM, and AS backbones, we were able to
produce a rough structural model of the catalytic domain of NPPs (Fig.
3B). A striking feature of this model is that the
metal-binding and active site residues in NPP1 are superposed onto
those of AP and iPGM. Thus, the 6 residues in APs and iPGMs that are
known to coordinate the two metals in their catalytic site are
conserved in the NPPs (Figs. 3A and 4) and were predicted to
display a similar spatial arrangement relative to the residue that
forms the catalytic intermediate, i.e. a Thr in NPPs (6, 42,
43) and a Ser in AP and iPGM (32, 44, 45). Arylsulfatases have a
similar catalytic core, but differ from AP and iPGMs in that they have
only a single metal-binding site and that the residue that forms the
catalytic intermediate is an oxidated cysteine (46).
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Fig. 3.
Structural similarities between the catalytic
domain of NPPs, APs, iPGMs, and
ASs. Panel A, the
consensus secondary structure of the catalytic domain of mouse NPP1
(residues 192-568) was predicted by combination of the PSI-PRED, PHD,
SamT99, JPRED, and Prof secondary-structure prediction algorithms. This
predicted structure was aligned with the known secondary structure of
the E. coli AP (PDB code 1ALK), the B. stearothermophilus iPGM (PDB code 1EJJ), and the human AS-A (PDB
code 1AUK), and AS-B (PDB code 1FSU), as determined with the
Swiss-PdbViewer (41). The regular numbers
indicate the distances to the beginning and the end of each protein,
and the numbers in parentheses indicate the sizes
of the gaps between the aligned segments. The superscript
numbers show the position of some key residues in each
protein. -Strands are shown in gray boxes and
are numbered, while the
-helices are in a
black box and are lettered.
,
Catalytic site residue,
, residues coordinating Zn2+
(Zn1) in AP and Mn2+ (Mn1) in iPGM;
residues
coordinating Zn2+ (Zn2) in AP and Mn2+ (Mn2) in
iPGM, Mg2+ in AS-A and Ca2+ in AS-B;
,
residue coordinating Mg2+ in AP. Panel
B shows a model of the catalytic core of NPPs, as deduced
from the conserved fold elements and known structure of APs, iPGMs, and
ASs. The
-helices and
-strands are numbered and lettered as in
panel A. The position of the active site threonine and of the residues
involved in the binding of metals (Me) are denoted as in
panel A. Points in the surface loops where
additional protein fragments should be inserted are indicated by
breaks. For none of the aligned proteins did the used set of
secondary prediction programs (see above) forecast an
-helical
structure for helix B. However, since this fragment is known to adopt
an
-helical structure in APs, iPGMs, and ASs, it was assumed that
the corresponding fragment of NPP1 also folds into an
-helix.
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Fig. 4.
Model of the catalytic core of NPPs. The
primary structure of the catalytic domain of mouse NPP1 was threaded
onto the known three-dimensional structure of the catalytic domain of
alkaline phosphatase (PDB code 1ALK). Using the known (AP) and
predicted (NPP) secondary structure as criteria (see Fig.
3A), the six Zn2+-coordinating residues of AP
showed a superposition with identical residues in all NPPs. In
addition, the catalytic site Thr of NPP1 coincided with that of the
catalytic site Ser of AP. The figure shows the predicted constellation
of the metal-binding residues and the catalytic site Thr of mouse NPP1.
A similar active site constellation has been derived from the crystal
structure of AS-A, AS-B, and iPGM (30-32). Red, oxygen;
blue, nitrogen; green, metal
(Me).
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Fig. 5.
Proposed reaction mechanism for NPPs.
The proposal is based on the known reaction mechanism of APs (44) and
iPGMs (32), and the conserved catalytic site structure elements of
NPPs, including the residues that coordinate Me1 and Me2 (Figs. 3 and
4). The open circles represent OH- or O-groups.
R refers to various structures, including a nucleoside
monophosphate (e.g. in AP2A), a phosphate
(e.g. in ADP), or a pyrophosphate (e.g. in ATP).
In the free enzyme (E), the hydroxyl group of the catalytic
site threonine (Thr-238 in mouse NPP1) is stabilized by Me2 in its
nucleophilic state. The enzyme-substrate complex
(E·R-O-NMP) is formed as a result of the
coordination of the NMP-ester oxygen atoms by Me1 and additional
interaction of one of the nonbridging oxygen atoms of the substrate
with Me2. Thr-238 (T238) holds a position opposite to the
leaving group of the NMP-ester substrate. A nucleophilic attack of the
hydroxyl group of Thr-238 results in the formation of the covalent,
nucleotidylated intermediate (E-NMP), departure of the
alcohol leaving-group and inversion of the phosphorus center. At
alkaline pH a water molecule coordinated by Me1 attacks the phosphorus
apically, resulting in the hydrolysis of the nucleotidylated
intermediate, inversion of the phosphorus center, and the formation of
a noncovalent enzyme-NMP complex (E·NMP). The
free enzyme is regenerated by dissociation of NMP (nucleoside
monophosphate). Also shown are the residues that coordinate Me1 and Me2
(see also Fig. 4).
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[in a new window]
Fig. 6.
Mutations affecting the NPP activity and/or
the formation of the nucleotidylated intermediate of NPP1. Mouse
NPP1 or the indicated mutants were expressed in COS-1 cells as fusions
with an HA tag. The fusion proteins were immunoprecipitated from the
cell lysates with anti-HA antibodies. Similar amounts of
immunoprecipitated fusion proteins, as quantified by Western analysis
with antibodies against the C terminus of NPP1 (panel
A), were used for the assay of phosphodiesterase activity
with p-nitrophenyl thymidine 5'-monophosphate as substrate,
in the absence (light gray bars) or
presence (dark gray bars) of 2 mM ZnCl2 (panel B). The
immunoprecipitated NPP1 fusions were also used for the trapping of the
nucleotidylated intermediate with 50 µM
[ -32P]ATP as substrate (panel
C). The nucleotidylated intermediate was visualized by
autoradiography after SDS-PAGE. The results in B represent
the means ± S.E. of eight to ten assays. The anti-HA antibodies
did not precipitate detectable amounts of NPP activity from cells that
had not been transfected with the NPP1 expression vector (data not
shown). WT, wild type.
-32P]ATP as a substrate (Fig.
6C). As expected, the inactive T238A had lost the ability to
form the nucleotidylated intermediate. In contrast, the poorly active
T238S mutant accumulated the nucleotidylated intermediate, which
indicates that the hydrolysis of the intermediate was hampered. A
similar reasoning applies to the D358N and H362Q mutants. An increased
labeling of the latter mutants could be expected since Asp-358 and
His-362 were proposed to be involved in the binding of Me1, which is
predicted to play an essential role in the second step of catalysis
(Fig. 5). The inability of the H517Q mutant to form the covalent
intermediate may suggest that the mutation of His-517 to a glutamine,
in addition to abolishing the binding of Me1, also disturbs the
coordination of Me2. Indeed, the mutation of any residue predicted to
be involved in the coordination of Me2 (D200N, D405N, H406Q) abolished
the formation of the nucleotidylated intermediate, in agreement with
the requirement of Me2 to start the catalytic cycle (Fig. 5). The F239A
mutant, which displayed about half of the wild type NPP activity (Fig.
6B), also showed a decreased ability to trap the catalytic
intermediate (Fig. 6C). Phe-239 is conserved in all NPPs but
not in APs, iPGMs, and ASs, indicating that the role of this residue is
specific for NPP substrates. For example, Phe-239 could be involved in
the coordination of the substrate. If so, the decreased activity and
level of covalent intermediate of the F239Q mutant may be accounted for
by a decreased binding of the substrate.
-32P]ADP (Fig.
7), establishing that it does not
represent a classical kinase reaction. Second, under identical
conditions, a labeling was also observed with AP from E. coli (data not shown) and from bovine intestinal mucose (Fig. 7),
showing that ATP and ADP are indeed substrates for APs. On the other
hand, neither NPP1 nor AP were labeled by incubation with
[32P]Pi, indicating that the labeling was not
due to the generation of Pi by hydrolysis of ATP or
ADP.
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[in a new window]
Fig. 7.
Autophosphorylation of NPP1 and AP.
Mouse NPP1, immunoprecipitated as an HA-tagged fusion protein from
COS-1 cell lysates, and bovine AP were incubated in the presence of 50 µM amounts of the indicated nucleotides
([ -32P]ATP (A) and
[
-32P]ADP (B)) or [32P]
Pi (C), as detailed under "Materials and
Methods." At the indicated time points, samples were taken for
Tricine-SDS-PAGE (7.5%) followed by autoradiography.
-32P]ATP. This
suggests strongly that the same catalytic site mediates both the
nucleotide pyrophosphatase/phosphodiesterase and the phosphatase
activities. Moreover, as was observed for the phosphodiesterase reaction (Fig. 6C), the T238S mutant could still
autophosphorylate (Fig. 8). Although the F239A mutant showed a reduced
capacity for nucleotidylation (Fig. 6C), its ability to form
a phosphorylated intermediate was not affected (Fig. 8). However, if
Phe-239 is involved in the coordination of the nucleoside moiety, as
suggested above, it can be expected that its mutation decreases the
phosphodiesterase activity without affecting the phosphatase activity
since both enzymic reactions require the substrate to be bound in a
different way (see also "Conclusions and Perspectives").
Unexpectedly, although the D358N and H362Q mutants showed an increased
labeling with [
-32P]ATP (Fig. 6C), the
labeling with [
-32P]ATP was decreased (Fig. 8). These
data suggest that Asp-358 and His-362, in addition to their role in
coordinating Me1, may also play a role in the binding of nucleotides as
substrates for the phosphatase reaction.
View larger version (27K):
[in a new window]
Fig. 8.
Mutations affecting the autophosphorylation
of NPP1. The same immunoprecipitated HA-tagged native and mutant
NPP1s (Fig. 6) were also used for labeling with 50 µM
[ -32P]ATP for 90 min at 30 °C, as detailed under
"Materials and Methods." The labeled HA-NPP1 fusions were
visualized by autoradiography after SDS-PAGE.
- or the
-phosphate that will become covalently bound to the catalytic site
threonine in the pyrophosphatase or phosphatase reaction, respectively.
Future investigations should provide a deeper insight into the binding
modes of the substrates and into the relative importance of the
nucleotide pyrophosphatase/phosphodiesterase and the phosphatase
reactions that are catalyzed by NPPs.
![]() |
ACKNOWLEDGEMENTS |
---|
Karolien Nelissen provided expert technical assistance. We acknowledge Monique Beullens for the preparation of NPP1 antibodies.
![]() |
FOOTNOTES |
---|
* This work was supported by the Fund for Scientific Research-Flanders Grant G.0237.98.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF233377.
To whom correspondence should be addressed: Afdeling Biochemie,
Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.:
32-16-34-57-01; Fax: 32-16-34-59-95; E-mail:
mathieu.bollen@med.kuleuven.ac.be.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007552200
2 The NPP nomenclature used here stems from a broad consensus at a recent international workshop on "Ecto-ATPases and Related Ectonucleotidases" (7).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NPP, nucleotide pyrophosphatase/phosphodiesterase; AP, alkaline phosphatase; AS, arylsulfatase; HA, hemagglutinin; iPGM, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; NCBI, National Center for Biotechnology Information; PDB, (Brookhaven) Protein Data Bank; PSI-BLAST, Position Specific Iteration Basic Local Alignment Search Tool; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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