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J Biol Chem, Vol. 274, Issue 50, 35434-35440, December 10, 1999
,From the Inositide Signaling Group, Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Diphosphoinositol polyphosphate phosphohydrolase
(DIPP) hydrolyzes diadenosine
5',5 The mammalian diphosphoinositol
polyphosphate phosphohydrolase
(DIPP)1 was originally
characterized (1) as being responsible for the dephosphorylation of
PP-InsP5 and (PP)2-InsP4 (Fig. 1),
which are the most highly phosphorylated members of the inositide
signaling family of molecules (2). PP-InsP5 and
(PP)2-InsP4 are widely distributed across the
phylogenetic spectrum; they have been identified in mammals (3, 4),
yeasts (5), slime molds (6-8), plants (9), and free living amoebae
(10, 11). The dephosphorylation of PP-InsP5 and
(PP)2-InsP4 results in a considerable free
energy change, in no small part due to the relief of the severe
electrostatic and steric constraints imposed by the high density of
phosphate groups that are clustered around the inositol ring (7).
Nevertheless, in vivo there is rapid turnover of both
PP-InsP5 and (PP)2-InsP4, so the
levels of these compounds must be continually replenished at a robust
rate. This ATP-consuming process therefore places a substantial burden
upon the cell's energy reserves (12). These observations have led to
the suggestion that PP-InsP5 and
(PP)2-InsP4 turnover might act as a molecular
switch for a very dynamic cellular process, for which vesicle
trafficking has emerged as a candidate (13, 14). Support for the idea
that PP-InsP5 in particular might regulate the movement of
vesicles through the cell comes from demonstrations that some of the
proteins that regulate this process bind PP-InsP5 very
tightly; examples include coatomer (5, 15), AP-2 (see Ref. 3), and AP-3
(16). Another energy-consuming activity that may be nominated for being
regulated by diphosphoinositol polyphosphates is mRNA export from
the nucleus. Recent studies indicate that efficient mRNA export in
yeasts requires the synthesis of inositol hexakisphosphate (17).
However, rather than inositol hexakisphosphate participating in this
process directly, as was originally proposed (17), the significance of
inositol hexakis- phosphate may actually be as the precursor of
PP-InsP5 and (PP)2-InsP4 (12), the
turnover of which can more readily account for some of the
energy-dependent aspects of mRNA export.
The contention that it is the actual turnover of diphosphoinositol
polyphosphates that is functionally significant is further supported by
their cellular levels being regulated by specific cell-signaling
events. These include the mobilization of certain categories of
cellular Ca2+ stores, which inhibits the synthesis of
PP-InsP5 (18). In addition, the metabolism of
diphosphoinositol polyphosphates is regulated by receptor-mediated
changes in levels of cAMP and cGMP (19). Since DIPP is the enzyme with
primary responsibility for controlling the dephosphorylation of
PP-InsP5 and (PP)2-InsP4 (1), this phosphohydrolase is a prime candidate for being regulated by these intracellular signaling processes.
Recently, the prominence of
DIPP2 was further enhanced by
the discovery that this enzyme also actively hydrolyzes diadenosine polyphosphates (20), particularly Ap6A (Fig. 1). This group of molecules act extracellularly as neurotransmitters (21) and vasomodulators (22, 23). Intracellularly, they are thought to regulate
cardiac KATP channels (24) and ryanodine
receptors (25). Clearly, enzymes that regulate the concentrations of
Ap6A in vivo can play pivotal roles in some
important signal transduction processes.
Given the importance of the two different catalytic activities of DIPP,
it is of considerable enzymological significance to understand how this
enzyme has the flexibility to hydrolyze both diadenosine polyphosphates
and the structurally unrelated diphosphoinositol polyphosphates (Fig.
1). There would appear to be little scope for two separate catalytic
centers in DIPP, considering its relatively small size of only 19.5 kDa
(1). In fact, a single candidate catalytic core in DIPP has been
proposed (1), on the basis of it matching a consensus for the catalytic
motif of Nudix hydrolases (nucleoside
diphosphate attached to another moiety, "x"
(26)). The Nudix designation embraces Ap6A, but not
PP-InsP5 and (PP)2-InsP4. Thus, one
of the goals of the current study was to ascertain whether amino acid
residues within the Nudt (Nudix-type) catalytic
core of DIPP were recruited for the hydrolysis of both classes of
substrates. Yet we also queried whether there might be some
substrate-dependent interactions with specific amino acid
residues, in part because DIPP cleaves the internal polyphosphate chain
of Ap6A, but the enzyme removes the terminal 
-P1,P6-hexaphosphate
(Ap6A), a Nudix (nucleoside
diphosphate attached-moiety "x") substrate,
and two non-Nudix compounds: diphosphoinositol pentakisphosphate
(PP-InsP5) and bis-diphosphoinositol
tetrakisphosphate ((PP)2-InsP4). Guided by
multiple sequence alignments, we used site-directed mutagenesis to
obtain new information concerning catalytically essential amino acid
residues in DIPP. Mutagenesis of either of two conserved glutamate
residues (Glu66 and Glu70) within the Nudt
(Nudix-type) catalytic motif impaired
hydrolysis of Ap6A, PP-InsP5, and
(PP)2-InsP4 >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains
(glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with
potential sites for cation coordination and substrate binding. GR1
comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX2GX6G) that is
conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly
residues in GR1 and GR2 reduced catalytic activity toward all three
substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y
mutations substantially decreased the rate of Ap6A and
(PP)2-InsP4 metabolism (by 71 and 96%), yet
PP-InsP5 hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His91
and Phe84. This new information helps define DIPP's
structural, functional, and evolutionary relationships to Nudix hydrolases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-phosphates
from PP-InsP5 and (PP)2-InsP4 (Fig.
1). Still further catalytic agility of DIPP must be accounted for
because of its alternative positional specificity when either PP-InsP5 or (PP)2-InsP4 is a
substrate (Fig. 1). It is the
5--phosphate that is cleaved from PP-InsP5 (3, 4), yet
the 5--phosphate on (PP)2-InsP4 is
apparently protected, and it is the other -phosphate that is
predominantly hydrolyzed (3).
View larger version (21K):
[in a new window]
Fig. 1.
The structures of Ap6A and the
mammalian forms of PP-InsP5 and
(PP)2-InsP4 and the sites of their hydrolysis
by DIPP. The structures of PP-InsP5,
(PP)2-InsP4, and Ap6A are shown.
The diphosphate group in mammalian PP-InsP5 has been
determined to be attached to the 5-carbon (4). Since
5-PP-InsP5 is the precursor of
(PP)2-InsP4, the latter also has a diphosphate
group in the 5-position. However, the location of the second
diphosphate group on (PP)2-InsP4 has not been
determined for any mammalian system; it is tentatively placed at the
6-position in the figure, since 5,6-(PP)2-InsP4
has been identified in Dictyostelids (8, 43). The solid
arrows indicate the major sites of attack upon these
substrates by DIPP. While Ap6A is predominantly converted
to AMP and adenosine 5'-pentaphosphate, lesser quantities of ADP plus
adenosine 5'-tetraphosphate are also formed (20); the latter is
schematically depicted by the unfilled
arrow.
To identify candidate active-site residues in DIPP, we first compared
the amino acid sequence of this enzyme with those of Aps1 and YOR163w,
which are two yeast proteins that share DIPP's ability to actively
hydrolyze diadenosine polyphosphates and diphosphoinositol polyphosphates (20). The sequences of these three proteins diverge considerably, except for a limited degree of conservation within the
Nudt motif and short flanking regions (20). Thus, this homologous region of DIPP was the area of focus for this study; we compared this
portion of DIPP with the corresponding sequences of the Nudix hydrolase
family members. In this paper, we outline how this alignment procedure
identified a subset of proteins with a number of conserved residues,
and we go on to describe how we obtained new information concerning
their functional significance, in the most detailed mutagenic study of
a Nudt protein described to date.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Materials-- PP-[3H]InsP5 was obtained from NEN Life Science Products; (PP)2-[3H]InsP4 was prepared as described previously (20). Ap6A was purchased from Sigma. The Partisphere 5-µm SAX column was purchased from Krackler Scientific (Durham, NC). NuPAGE gels were obtained from Novex (San Diego, CA).
Site-directed Mutagenesis of Recombinant DIPP-- Wild-type, recombinant human DIPP was prepared as described previously (1). The recombinant DIPP mutants were prepared by using the QuickChangeTM site-directed mutagenesis kit from Stratagene, according to the manufacturer's instructions. Two complementary mutagenic primers, each containing one base mismatch, were synthesized with a Beckman Oligo 1000M DNA synthesizer (Table I); the preparation of the E70Q mutant was described previously (1). The cDNA construct for recombinant DIPP was used as template for PCR using the mutagenic primers. The wild-type strand in the resulting PCR product was removed by digestion with 10 units of DpnI at 37 °C for 1 h before being used to transform Escherichia coli XL1-Blue supercompetent cells. The colonies from the transformation plates were screened by colony PCR with two primers flanking the DIPP gene, and plasmids were prepared from the colonies that generated the expected PCR product. The presence of the correct mutation in all of the constructs was verified by DNA sequencing using a PCR-based dRhodamine fluorescent dye method (Perkin-Elmer).
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Expression and Purification of Recombinant DIPP and Its
Mutants--
The cDNAs for DIPP and each of the mutants were
transformed into M15 competent cells, which were incubated overnight at
37 °C in 10 ml of LB broth containing 100 µg/ml of ampicillin and 25 µg/ml of kanamycin. The overnight culture was inoculated into 100 ml of the same medium, and the incubation was continued until A600 reached 0.9-1.0, whereupon 1.5 mM isopropyl--thiogalactopyranoside was added to
initiate the expression. After an additional 5 h, the cells were
harvested by centrifugation. The techniques used to lyse the cells and
to purify recombinant DIPP and the various mutants using a
nickel-nitrilotriacetic acid Superflow column are all as described
previously (1). To increase the quantity of protein for analysis by
circular dichroism, 10 ml of the overnight culture was inoculated into
250 ml of LB medium in the expression step. In addition, the peak
fraction eluted from the nickel-nitrilotriacetic acid column was placed
in a 3-ml Slide-A-Lyzer cassette (Pierce) and dialyzed against 4 liters
of 10 mM phosphate buffer (pH 7.5). The dialyzed protein
was then filtered (0.45 µm), and its protein concentration was
determined according to Beer's law. The extinction coefficient at 280 nm was calculated (27) to be 27,670 M
1
cm1 for recombinant DIPP and its mutants (except F84Y,
which was 28,950 M1 cm1). The
final concentration of the dialyzed mutants was between 9.1 and 32.2 mM. The purity of each protein preparation was determined on 4-12% gradient NuPAGE gels (Fig. 2)
with MES SDS running buffer.
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Structural Analysis by Circular Dichroism
Spectroscopy--
Far-ultraviolet circular dichroism spectra were
acquired on a Jasco J-600 spectropolarimeter equipped with a
thermostatically regulated cuvette (set to 15 °C) with a 1-mm path
length, using a resolution of 0.1 nm, a bandwidth of 1.0 nm, and a
response time of 2 s. Samples were diluted to 9.1-11.2
mM of protein in 10 mM sodium phosphate buffer,
pH 7.5. Five CD spectra were acquired and averaged before the buffer
background was subtracted. Estimates for 
-helix and -pleated
sheet content were obtained using the Selcon fitting program.
Enzyme Assays--
First-order rate constants for the
dephosphorylation of PP-[3H]InsP5 and
(PP)2-[3H]InsP4 were determined
as described previously (1). The dephosphorylation of 100 µM Ap6A was measured in 100-µl assays (20)
that were quenched with 50 µl of ice-cold 2 M
trichloracetic acid. Samples were then neutralized with 100 µl of 1 M K2CO3, 5 mM EDTA.
Reaction products were resolved by high pressure liquid chromatography using a 4.6 × 125-mm Partisphere 5-µm SAX column, eluted at 1 ml/min by the following gradient generated by mixing buffer A (1 mM Na2EDTA) and buffer B (buffer A plus 1.3 M (NH4)2HPO4, pH 3.85, with H3PO4): 0-1 min, 0% B; 1-31 min,
0-25% B; 31-51 min, 25-55% B; 52-62 min, 0% B. Metabolites were
identified as described previously (28).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Identification of Candidate Functional Elements of DIPP by Multiple
Sequence Alignments--
Mammalian DIPP has dual specificity, in that
it hydrolyzes diphosphoinositol polyphosphates (1) and diadenosine
polyphosphates (20). Two yeast proteins (Aps1 and YOR163w; see Ref. 20
and Fig. 3) have been identified that
also hydrolyze these two different groups of substrates. The amino acid
sequences of these yeast proteins diverge considerably from that of
DIPP, except for a limited degree of conservation within the central
core that includes the Nudt catalytic motif and short flanking regions
(20). We focused on this homologous internal region of DIPP, and
aligned it with a large number of Nudix hydrolases, including 40 that were listed in a recent study (29). A small subgroup of these proteins
were identified, in which there are varying degrees of conservation of
several interesting features (Fig. 3): (i) highly conserved glutamate
residues within the Nudt motif; (ii) Gly-rich regions flanking the Nudt
motif; (iii) a conserved phenylalanine residue; and (iv) a patch of
amino acids with strongly positive electrostatic potential, in which a
His residue is the most strongly conserved.
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The identification of these conserved elements forms the basis for the mutagenic strategy that we have used in this study.
Glutamate Residues within the Nudt Motif Are Essential for the Hydrolysis of Diphosphoinositol Polyphosphates and Diadenosine Polyphosphates-- In our sequence alignments (Fig. 3), five of the seven Glu residues in the Nudt region of DIPP are highly conserved; two of these aligned Glu residues have previously been shown to be catalytically essential in other Nudt contexts (Glu52 in human MutT homologue type 1 (30) and Glu57 in MutT (31)). We therefore targeted the equivalent Glu residues in DIPP (Glu66 and Glu70; see Fig. 3).
The E70Q mutant of recombinant DIPP had a greatly reduced rate of
hydrolysis of PP-InsP5 and
(PP)2-InsP4 (Fig.
4 and Ref. 1). We have extended this
observation by demonstrating that this same E70Q mutation also greatly
impaired (by 98%) the ability of recombinant DIPP to hydrolyze
Ap6A (Fig. 4). The E66Q recombinant DIPP mutant also had no
significant activity toward either
PP-[3H]InsP5 or
(PP)2-[3H]InsP4, and hydrolase
activity against Ap6A was found to be only 2% as active as
the wild-type enzyme (Fig. 4). The catalytic paradigm for the Nudt
motif envisages these two Glu residues participating in both binding of
the Mg2+-substrate complex and in general base catalysis
(31, 32). Our experiments are consistent with this catalytic process in DIPP being utilized for the hydrolysis of both diphosphoinositol polyphosphates and diadenosine polyphosphates.
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We also used CD spectroscopy to examine whether these two mutations had
any effect on the secondary structure of recombinant DIPP. The CD
spectra of the E66Q and E70Q mutants were not significantly different
from that of wild-type recombinant DIPP (data not shown). The CD
spectrum for the wild-type enzyme (see below), when analyzed using the
Selcon computer algorithm, indicated that the content of -helix and
-sheet was 18 ± 0.3 and 29 ± 1.2%, respectively (n = 4).
Substrate Recognition outside the Nudt Domain: Roles of Phe84 and His91-- Although the Nudt motif is well recognized to represent the catalytic core of the family of proteins in which it occurs (26), relatively little is known of the contributions to catalysis made by the amino acid residues that lie outside this conserved motif. Previously published sequence alignments of Nudix hydrolases have identified a conserved Phe residue that lies downstream of the eponymous catalytic motif (33); this residue is believed to ensure appropriate positioning of the nucleotide ring (31, 32), although the impact of mutagenesis of this residue has not previously been studied. Our own multiple sequence alignments suggested that this conserved residue was equivalent to Phe84 in DIPP (Fig. 3). Thus, an F84Y mutant was constructed to further examine this idea. The CD spectra indicated that this mutation did not have a significant effect on the secondary structure of the protein (data not shown). However, the rates of hydrolysis of (PP)2-InsP4 and Ap6A were substantially impaired (by 63 and 75%, respectively; Fig. 4). In contrast, the rate of hydrolysis of PP-InsP5 was only slightly reduced in the F84Y recombinant DIPP mutant (by only 30%; Fig. 4), suggesting only a relatively minor role for Phe84 in the dephosphorylation of that particular substrate.
A recurring characteristic to emerge from our multiple sequence
alignment (Fig. 3) is the presence, in six of these eight proteins, of
a patch of between two and five residues with strong positive
electrostatic potential, beginning 16-23 residues downstream of the
carboxyl terminus of the Nudt motif. This kernel of positive charge is
a good candidate for having electrostatic interactions with the
negative charges carried by the phosphate groups on DIPP's substrates.
The most conserved of this group of residues (His91) was
chosen as a target for further study of this particular issue. An H91L
mutant of recombinant DIPP was constructed; CD spectra indicated that
this amino acid substitution had no significant effect on the overall
secondary structure (data not shown). With PP-InsP5 as
substrate, the H91L mutant expressed nearly normal phosphohydrolase
activity (70% of the activity of the wild type recombinant DIPP; see
Fig. 4). The hydrolysis of Ap6A was more substantially
impaired in the H91L mutant (26% of the activity of the wild type
enzyme; Fig. 4). However, the most profound effect of this mutation was
observed with (PP)2-InsP4 as substrate; the H91L mutant expressed only 4% of the activity of the wild-type enzyme
(Fig. 4). The particular sensitivity of
(PP)2-InsP4 hydrolysis to the H91L mutation,
especially when compared with its relatively small impact upon
PP-InsP5 metabolism, may be relevant to the differential
positional specificity of DIPP toward these two substrates (Fig. 1);
i.e. the 5--phosphate is hydrolyzed from
PP-InsP5 (3, 4), but this same phosphate group on
(PP)2-InsP4 is apparently protected from
attack, and it is the other -phosphate that is hydrolyzed (3);
His91 appears to have relatively specific interactions with
(PP)2-InsP4.
The Identification and Potential Significance of Conserved Glycine-rich Domains Flanking the Nudt Motif of DIPP-- DIPP contains two short Gly-enriched regions (GR1 and GR2; see Fig. 3) that extend out from each end of the Nudt motif in DIPP. Despite their short length, these two regions together contain over half of DIPP's total complement of 13 Gly residues (1). Our sequence alignments (Fig. 3) reveal that three of the four Gly residues in the GR2 region comprise a consensus sequence (GX2GX6G) that is conserved in both Aps1 and YOR163w, which, like DIPP, also express dual specificity toward diphosphoinositol polyphosphates and diadenosine polyphosphates (20). As for GR1, this tripeptide is unique to DIPP in our alignments, but Gly itself is represented between two and four times in the first nine residues of all the sequences in Fig. 3.
One clue as to the significance of these Gly clusters comes from
predictive analyses of the general architecture of the Nudt motif,
which suggests an -helix flanked by loops (34, 35). This arrangement
was empirically confirmed by the determination of the solution
structure of the prototypical Nudix hydrolase, the E. coli
MutT protein that attacks 8-oxo-dGTP (32). The importance of Gly
residues in GR1 and GR2 may be to construct the tight bends in the
polypeptide backbone upon which the loop structure depends (36).
However, the significance of Gly can extend beyond this structural
role; there are circumstances in which these loops form compact
ligand-binding pockets in which Gly itself directly participates. Two
types of Gly-ligand interactions are suggested by the Nudix context in
which GR1 and GR2 are found. First, one of the binding sites for the
catalytically essential divalent cation in the E. coli MutT
protein is the carbonyl carbon of Gly38 (31, 32). This
residue is conserved in other Nudix hydrolases and corresponds to
Gly51 in the GR1 region of DIPP (Fig. 3). Second, since
Ap6A is a nucleotide, it is significant that Gly-rich
sequences are also a recurring feature of nucleotide-binding loops (36,
37).3 The absence of a side
chain in the Gly residue permits a phosphate group to approach the
backbone amides, to which the ligand is gripped by hydrogen bonds (38,
39). This represents a potential role for GR2.
GR1: Mutagenesis of Gly50, Gly51, and
Gly52--
Our proposition that Gly51 may be
functionally significant in GR1 (see above) was pursued by
conservatively mutating this residue to Ala. This mutant of recombinant
DIPP had the same CD spectrum as the wild-type enzyme (data not shown),
but its catalytic activity was severely compromised (>97% less than
that of the wild-type enzyme; see Fig.
5). Assuming we are correct with our
suggestion that the cation-binding function for the carbonyl carbon of
Gly38 in the E. coli MutT protein (31, 32) is
conserved for Gly51 in DIPP (see above), then we can
rationalize the effect of the G51A mutation; the steric bulk of the Ala
side chain may prevent the cation from approaching the backbone
carbonyl group of Gly51.
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We also examined the importance to catalysis of the two Gly residues that flank Gly51. The rates of hydrolysis of diphosphoinositol polyphosphates by G50A and G52A mutants of recombinant DIPP was substantially (>85%) less than that of the wild type enzyme (Fig. 5). The hydrolysis of Ap6A was also decreased compared with wild type recombinant DIPP, although slightly less dramatically (by 60-75%; see Fig. 5). The rate of Ap6A hydrolysis was further reduced (by 90-99%; see Fig. 5) in G50V and G52V mutants of recombinant DIPP. The fact that Gly-to-Val mutants are impaired more dramatically than Gly-to-Ala substitutions is again consistent with the idea that the introduction of steric bulk in the GR1 region prevents proper ligand-protein interactions. Circular dichroism spectra indicated that the secondary structure of these additional mutants were not significantly different from that of the wild type recombinant DIPP (data not shown).
GR2: Mutagenesis of Gly72, Gly75,
Gly78, and Gly82--
Four separate Gly-to-Ala
mutants of recombinant DIPP were constructed in the GR2 region: G72A,
G75A, G78A, and G82A. The catalytic activity of the G78A mutant (Fig.
6) and its CD spectrum (data not shown)
were not significantly different from that of the wild-type enzyme.
This is an interesting observation, because Gly78 is the
only one of these four Gly residues that is not conserved in Aps1 and
YOR163w. Note, however, that the additional steric bulk introduced in a
G78V mutant was not tolerated, and then there was a substantial
decrease in catalytic activity toward all three substrates (Fig.
6).
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The CD spectrum obtained from the G82A mutant showed a substantial
change from that of the wild-type enzyme (Fig.
7). The nadir in the spectrum at
203 nm broadened considerably and increased in value from
9000 to 3000 degrees·cm2·dmol1 (Fig.
7). The Selcon computer algorithm estimated that, compared with the
wild-type recombinant DIPP, the content of -helix in the G82A mutant
was 36% lower, while the -sheet content was 30% higher.
Considering the very conservative nature of the Gly-to-Ala substitution, the consequences for the overall secondary structure of
the protein are very dramatic. Further structural analysis will be
required in order to account for this effect. However, in view of the
fact that Gly plays an important role in the architecture of certain
polypeptide loops (36), such as those that generally flank the Nudt
motif (31, 32, 34, 35), it is notable that there are examples of
proteins where no other amino acid can replace Gly at particularly
tight turns in a loop (40); this may be why the introduction of an G82A
mutation promotes some unraveling of the normal secondary structure.
Presumably as a consequence, the catalytic activity of this mutant
recombinant DIPP is substantially impaired (Fig. 6).
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The CD spectra of the remaining two mutants, G72A and G75A, were both very similar to that of the wild-type enzyme (data not shown), indicating that the mutations had no significant impact upon the secondary structure. However, the catalytic activities of the G72A and G75A mutants toward PP-InsP5, (PP)2-InsP4, and Ap6A were impaired by >95% (Fig. 6). Our mutagenesis of the GR2 region of DIPP has therefore identified a novel Gly-rich consensus, GX2GX6G, that is essential for this enzyme's dual specificity.
General Conclusions-- The importance of this study primarily lies in its providing new information relevant to our understanding of the structural and functional relationship that DIPP has with Nudix hydrolases. This knowledge is particularly significant, because the metabolism of diphosphoinositol polyphosphates is an unorthodox activity for a protein that has a Nudt catalytic motif. Our study also breaks new ground by identifying some catalytically important residues that lie outside the Nudt consensus. It is these extramural residues that are believed to impart substrate specificity upon each of the individual members of this hydrolase family (26). We have found several residues in this category that make important contributions to the unusual dual catalytic specificity of DIPP.
A particularly striking illustration of these developments is our identification of a new functionally and structurally important array of Gly residues (the "GR2" motif, GX2GX6G) that projects out from the carboxyl terminus of the Nudt domain. The GR2 motif is conserved in Aps1 and YOR163w (Fig. 3), which are two yeast homologues of DIPP that also express dual specificity toward diadenosine polyphosphates and diphosphoinositol polyphosphates (20). Moreover, this GR2 motif is absent from two Nudix hydrolases that actively degrade diadenosine polyphosphates but do not attack diphosphoinositol polyphosphates, namely human APAH1 (20, 35) and the IalA invasion protein of Bartonella bacilliformis (20, 41, 42). These comparisons suggest that the emergence of the GR2 motif has been an essential factor for the evolution of dual specificity toward diadenosine polyphosphates and diphosphoinositol phosphates. In this case, the GR2 motif may be a signature sequence that helps predict which other proteins with a Nudt motif may have a similar catalytic activity. We have already identified one candidate: the putative sll1058 protein from Cyanobacterium synechocystis (Fig. 3).
Among other catalytically essential features of DIPP that we identified
in this study, His91 was found to be of special
significance; the mutation of this residue to Leu inhibited
(PP)2-InsP4 dephosphorylation by 96%, yet
PP-InsP5 metabolism was only reduced by 30% (Fig. 4). One other residue, Phe84, was also found to contribute more to
the hydrolysis of (PP)2-InsP4 compared with
PP-InsP5 (Fig. 4). These findings provide the first evidence for there being a substantial difference in active-site residues that participate in the hydrolysis of these two substrates. As
such, this is an important step forward toward the goal of understanding how DIPP has the flexibility to express differential positional specificity toward these two inositol-based substrates (see
Fig. 1).
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ACKNOWLEDGEMENTS |
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We are indebted to Devon Allen for expert assistance with the CD measurements. We also thank Drs. Richard McKay and Kiyoshi Itagaki for helpful comments during the writing of this manuscript.
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FOOTNOTES |
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* 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: Inositide Signaling
Group, Laboratory of Signal Transduction, NIEHS, National Institutes of
Health, Research Triangle Park, P.O. Box 12233, NC 27709. Tel.
919-541-3308; Fax: 919-541-0559; E-mail: yang3@niehs.nih.gov.
2 The continued use of DIPP as an acronym to describe this enzyme does not prejudge whether diphosphoinositol polyphosphates or diadenosine polyphosphates are the favored substrates in vivo in all specialized subcellular microenvironments.
3 Some of these sequences are known as "P-loops" (37), but these represent only a subset of a wider collection of Gly-rich, phosphate-binding sequences (36, 39).
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ABBREVIATIONS |
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The abbreviations used are:
DIPP, diphosphoinositol polyphosphate phosphohydrolase;
Ap6A, diadenosine 5',5-P1,P6-hexaphosphate;
Aps1, Ap6A hydrolase;
PP-InsP5, diphosphoinositol
pentakisphosphate;
(PP)2-InsP4, bis-diphosphoinositol tetrakisphosphate;
PCR, polymerase
chain reaction;
MES, 4-morpholineethanesulfonic acid;
Nudix, nucleoside
diphosphate attached to another moiety, "x";
Nudt, Nudix-type (the
Human Genome Nomenclature Committee recently implemented "NUDT" as
an acronym for the gene family that produces proteins that contain the
Nudix recognition motif, even if Nudix hydrolase activity is not the
primary function of the protein).
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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