From the Laboratory of Physiological Chemistry, Christian de Duve
Institute of Cellular Pathology and Catholic University of Louvain,
B-1200 Brussels, Belgium and the Ludwig Institute of
Cancer Research, B-1200 Brussels, Belgium
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ABSTRACT |
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When incubated with their substrates, human
phosphomannomutase and L-3-phosphoserine phosphatase
are known to form phosphoenzymes with chemical characteristics of an
acyl-phosphate. The phosphorylated residue in phosphomannomutase has
now been identified by mass spectrometry after reduction of the
phosphoenzyme with tritiated borohydride and trypsin digestion. It is
the first aspartate in a conserved DVDGT motif. Replacement of either
aspartate of this motif by asparagine or glutamate resulted in complete
inactivation of the enzyme. The same mutations performed in the
DXDST motif of L-3-phosphoserine phosphatase
also resulted in complete inactivation of the enzyme, except for the
replacement of the second aspartate by glutamate, which reduced the
activity by only about 40%. This suggests that the first aspartate of
the motif is also the phosphorylated residue in
L-3-phosphoserine phosphatase. Data banks contained seven
other phosphomutases or phosphatases sharing a similar, totally
conserved DXDX(T/V) motif at their amino
terminus. One of these (-phosphoglucomutase) is shown to form a
phosphoenzyme with the characteristics of an acyl-phosphate. In
conclusion, phosphomannomutase and L-3-phosphoserine
phosphatase belong to a new phosphotransferase family with an
amino-terminal DXDX(T/V) motif that serves as
an intermediate phosphoryl acceptor.
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INTRODUCTION |
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Phosphoryl transfer reactions occur through two distinct
mechanisms: (a) direct in-line transfer from donor to
acceptor via a ternary complex formed by the enzyme and its
two substrates and (b) two successive transfers with the
enzyme playing the role of an intermediate acceptor (reviewed in Ref.
1). Different amino acid side-chains are known to play the role of
acceptor in the second class of enzymes, i.e. serine in
alkaline phosphatase (2) and -phosphoglucomutase (3, 4); histidine
in phosphoglycerate mutase (5) and fructose 2,6-bisphosphatase (6);
cysteine in protein-tyrosine phosphatase (7) and aspartate in P-type ATPases (8).
The discovery of L-3-phosphoserine phosphatase (PSP)1 (9) and phosphomannomutase (PMM) (10) deficiencies led us recently to overexpress these enzymes and to investigate their properties (11-13). Quite remarkably, we found that both of them form, when incubated with their substrate, a phosphoenzyme that is extremely labile in the presence of alkali or hydroxylamine and that most likely corresponds to a phosphoaspartate or a phosphoglutamate residue.
In the present work, we provide evidence for the fact that the two enzymes are phosphorylated on the first aspartate in a common DXDXT motif that is conserved in both cases. We also show that this conserved motif is shared by several other phosphotransferases.
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EXPERIMENTAL PROCEDURES |
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Materials--
Glucose and Mes were from Acros (Geel, Belgium).
Other chemicals were from Sigma or Merck. [-32P]ATP,
T7 Thermosequenase, L-[14C]serine, and
[3H]NaBH4 were from Amersham Pharmacia
Biotech. Phosphomannose isomerase was from Sigma; other auxiliary
enzymes, Pwo polymerase, the restriction enzymes, and
NADP+ were from Boehringer. Trypsin, from Life
Technologies, Inc., had not been treated with
tosyl-L-phenylalanine chloromethylketone. Bacto tryptone
and Bacto yeast extract were from Difco Laboratories. Mannose
1,6-bisphosphate was prepared, purified, and titrated as in Ref. 13.
Dowex AG 1-X8 (200-400 mesh) was from Bio-Rad. L-3-[14C]Phosphoserine was prepared as
described in Ref. 14 and [32P]glucose 6-phosphate as
described in Ref. 15. Unless otherwise indicated, wild-type human PMM1
(12) and PSP (11) were produced and purified as described
previously.
Identification of the Phosphorylated Residue in Phosphomannomutase 1 by Reduction with [3H]NaBH4 and Mass Spectrometry-- Human recombinant PMM1 (50 µg) was incubated at 0 °C in a mixture (100 µl) containing 20 mM Hepes, pH 7.1, 1 µM mannose 6-phosphate, no (control) or 20 µM mannose 1,6-bisphosphate, 1 mM dithiothreitol, and 2 mM MgCl2. The reaction was stopped after 5 s by the addition of 500 µl of 10% ice-cold trichloroacetic acid. After centrifugation (20 min at 10 000 × g and 4 °C), the pellet was resuspended in 500 µl of 5% ice-cold trichloroacetic acid and recentrifuged for 10 min at 4 °C. The pellet was washed with 10 mM ice-cold HCl, centrifuged for 10 min at 4 °C, and dried under vacuum.
After resuspension of the pellet in 57 µl of Me2SO, 10 mCi of [3H]NaBH4 (5 Ci/mmol) in 10 µl of Me2SO and 2.7 µmol of unlabeled NaBH4 in 33 µl of Me2SO were successively added. After 10 min of incubation at 20 °C, 900 µl of 0.44 M ice-cold HClO4 was added; the sample was placed on ice for 30 min and then centrifuged for 20 min at 4 °C at 10 000 × g. The resulting pellet was washed with 500 µl of cold acetone and dried under vacuum. The protein was then resuspended in 200 µl of a mixture containing 0.1 M Tris/HCl, pH 8.6, 2 M urea, and 1 µg of trypsin; the mixture was incubated at 37 °C for 18 h. The reaction was stopped by the addition of 8 µl of 50% trifluoroacetic acid, and the mixture was applied onto an Alltech C18-reverse phase HPLC column (1 × 250 mm). Elution was accomplished with a linear aqueous 5-100% acetonitrile gradient in 0.1% trifluoroacetic acid over 100 min at a flow rate of 80 µl/min. Samples of the fractions were counted for radioactivity and analyzed by mass spectrometry. All mass spectral analyses were performed on a Finnigan LCQ ion-trap equipped with an electrospray source. The samples were introduced directly into the source at a flow rate of 2 µl/min. The spray was obtained by applying a potential difference of 5 kV and with the help of a sheath gas (N2) at 30 arbitrary units. The temperature of the heated capillary was 250 °C. The LCQ was operated under manual control in the Tune Plus view with default parameters and active automatic gain control.Site-directed Mutagenesis-- Site-directed mutagenesis was performed by using Pwo polymerase and "back-to-back" mutated primers as described in Ref. 16. The primers used to introduce the mutations corresponded to nucleotides 44 to 70 and 71 to 100 of the PMM1 sequence (EMBL U62526) and to nucleotides 224 to 259 and 260 to 295 of the human PSP cDNA (EMBL Y10275). The polymerase chain reaction was directly performed on a pET3d plasmid (17) containing the sequence of human PMM1 (12). The plasmids were recircularized, amplified in Escherichia coli JM109, and checked by sequencing with the T7 primer using T7 Thermo Sequenase (Amersham), fluorescent primers, and the LI-COR automated DNA sequencer 4000L. For the PSP mutants, the polymerase chain reaction was performed using a pBlueScript plasmid containing the coding sequence (11). The amplified plasmids were recircularized and introduced into E. coli JM109. Clones were then sequenced with the M13F primer to confirm the presence of the mutations and rule out polymerase chain reaction errors. NdeI-BamHI fragments were then excised and ligated into pET3a.
Expression of the Recombinant Proteins--
Bacteria
BL21(DE3)pLysS (17) harboring the expression plasmids were grown
aerobically in 0.5 liters of M9 medium at 37 °C until
A600 reached 0.5-0.6. The culture was then
maintained on ice for 60 min before addition of
isopropylthiogalactoside to a final concentration of 0.4 mM. For PMM mutants, incubation was carried out at 18 °C
for 13 h, whereas PSP was expressed during 20 h at 37 °C.
Bacterial extracts were prepared and the recombinant enzymes purified
by chromatography on DEAE-Sepharose as described in Ref. 11. The
wild-type PSP and the Asp22 Glu mutant of this enzyme
were further purified by chromatography on Sephacryl S-200 (11).
Purification and Labeling of -Phosphoglucomutase from
Lactococcus lactis--
L. lactis subsp. lactis
AS211 (kindly provided by P. Radström, Lünd, Sweden) was
grown anaerobically for 14 h at 37 °C in 3.5 liters of a medium
containing 10 g/liter Bacto tryptone, 10 g/liter Bacto yeast extract, 2 g/liter casaminoacids, 5 g/liter KH2PO4, 5 g/liter K2HPO4, 1 g/liter
MgSO4·7H2O, and 20 g/liter maltose. The
bacteria were collected by centrifugation at 5000 × g
and 4 °C for 15 min, resuspended in a buffer containing 50 mM Hepes, pH 7.1, 2.5 µg/ml leupeptin, 2.5 µg/ml
antipain, and 1 mM dithiothreitol and disrupted in a French
press. The homogenate was centrifuged at 10,000 × g
and 4 °C for 15 min, and the supernatant was applied onto a
DEAE-Sepharose column (1.5 × 15 cm) equilibrated in 50 mM Hepes, pH 7.1, 1 mM dithiothreitol. The
column was washed with 60 ml of this buffer, and proteins were eluted
with an NaCl gradient (0-750 mM in 250 ml of the same
buffer). The specific activity of the partially purified enzyme was
0.3 units/mg protein.
Computer Analysis-- The BLAST program from the NCBI (18) was used to search for homologous proteins in data banks. Multiple sequence alignments were performed with the PILEUP algorithm (Wisconsin Package version 8, Genetics Computer Group, Madison, WI).
Enzyme and Protein Assays-- PSP was assayed at 30 °C by the release of L-[14C]serine from L-3-[14C]phosphoserine in an assay mixture (100 µl) containing 25 mM Mes (pH 6.5), 1 mM MgCl2, 1 mM dithiothreitol, 0.1 mM L-3-phosphoserine for the human recombinant protein or 1 mM for the bacterial enzyme, 15,000 cpm of L-3-[14C]phosphoserine, 0.1 mg/ml bovine serum albumin. Reactions were stopped by addition of 100 µl of 10% HClO4. After centrifugation of the samples, the supernatants were neutralized with KHCO3 and processed as described in Ref. 14.
PMM was assayed as described (13) except that the pH was 7.5 for the bacterial enzyme and 6.5 for the human recombinant protein. ![]() |
RESULTS |
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Identification of the Phosphorylated Residue in Phosphomannomutase 1-- Phosphoaspartyl and phosphoglutamyl residues are too labile to resist the procedure commonly used to identify phosphorylated residues, which involves digestion of the phosphorylated protein with trypsin, separation of the resulting peptides by HPLC, and sequencing of the phosphorylated peptide. We therefore used the method of Degani and Boyer (20), in which the carboxyl-phosphate group is reduced with tritiated borohydride to a hydroxymethyl group before further processing of the protein. Radioactivity screening allows the detection of fractions containing the modified peptide. Furthermore, the replacement of a carboxyl group by a hydroxymethyl group results in a 14-dalton decrease of the molecular mass that allows the localization of the modified residue by mass spectrometry (21).
Human PMM1, incubated with (phosphorylated) or without (control) mannose 1,6-bisphosphate, was reduced by [3H]NaBH4 and submitted to digestion with trypsin. The peptides were separated by HPLC. As shown in Fig. 1, two peaks of radioactivity were found both with the control (C) and the phosphorylated (P) enzymes. The first one was significantly broader in the case of the column loaded with the phosphorylated reduced enzyme, due to the presence of much more radioactivity in fractions P-20 and P-21 than in the corresponding fractions (C-20 and C-21) of the control column. When analyzed by electrospray mass spectrometry, fraction P-21 was found to contain two peptides that differed by 14 amu and appeared in positive mode as [M+H]+ ions of m/z 1044.2 and 1030.2 in a 3:1 ratio (approximate) and in negative mode as ions of m/z 1042.3 and 1028.4 (not shown). Only the heavier of the two peptides was found in the corresponding fraction of the control column (C-21).
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Site-directed Mutagenesis of Phosphomannomutase 1-- To confirm the conclusion of the mass spectrometry analysis, we constructed PMM1 mutants in which we replaced separately the two aspartates of the conserved motif DVDGT by either glutamate or asparagine. Preliminary experiments indicated that the mutant proteins were insoluble when expressed at 37 °C. Expression was therefore carried out at 18 °C, not only for the mutated proteins but also for the human wild-type enzyme. Extracts prepared 13 h after addition of isopropylthiogalactoside to the culture contained activities (0.003-0.006 units/mg protein) that were much lower in the case of the mutated proteins than in that of the human wild-type enzyme (1.8 units/mg protein) and in fact comparable with the activity observed in a control culture without translatable insert (0.006 unit/mg protein).
All of these extracts were chromatographed on DEAE-Sepharose. When extracts containing the human wild-type enzyme were applied on the column, the elution profile of the PMM activity (Fig. 3A) coincided with that of the overexpressed 30-kDa polypeptide (not shown), as determined by SDS-PAGE analysis. When bacterial extracts containing the mutant proteins were chromatographed, the peak of PMM activity was about 500-fold lower, as in columns performed with control E. coli extracts. Furthermore, it did not correspond with the peak of recombinant protein, the amount of which was comparable with that of wild-type PMM. These results are exemplified for mutant Asp19
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Studies on L-3-Phosphoserine Phosphatase-- We attempted to determine the phosphorylation site of PSP by using the same approach as described above for PMM1. The enzyme was phosphorylated by incubation with a saturating concentration (20 mM) of its substrate, in the presence of 20 mM L-serine, to slow down the hydrolysis of the phosphoenzyme, precipitated with trichloroacetic acid, reduced with tritiated borohydride, and digested with trypsin. However, no distinct narrow peak of radioactivity could be found in the eluate of the column, presumably because of the low stoichiometry of phosphate incorporation into the enzyme (11).
We have previously noticed (11) that PSPs contain close to their amino terminus a conserved DXDST motif. As this motif is similar to the DVDGT sequence found in PMM, it was likely to contain the phosphorylation site of PSP. To investigate this possibility, we mutated the two aspartate residues of the DXDST motif to asparagine and glutamate, as described above for PMM1. Expression was carried out at 37 °C for 18 h. PSP activity was measured in cell extracts. For three of the four mutants (Asp20Sequence Comparisons--
The results mentioned above showed that
a region containing a DXDXT motif was important
for the function of PSP and of PMM. To find other enzymes with the same
or a related motif, we performed a BLAST search (18) in the GenBank and
EMBL data banks using the sequence of both enzymes, and we performed
additional searches with the sequences of the homologous enzymes that
were found. For all enzymes identified in this way, we aligned
sequences from different species to check the conservation of the
motifs. As shown in Fig. 4, several
enzymes were indeed found to possess a motif related to that found in
PMM and PSP. Several of these enzymes were previously identified (23)
as belonging to a large family of hydrolases comprising phosphatases
(PSP, trehalose 6-phosphate phosphatase, phosphoglycolate phosphatase,
histidinol phosphatase, and paranitrophenylphosphatase) as well as
2-L-haloacid dehalogenase and epoxide hydrolase. We added
to this list two mutases (PMM and -phosphoglucomutase), glycerol
3-phosphate phosphatase, and P-ATPases, and we removed epoxide
hydrolase because the amino-terminal motif (DLDGV) found in the human
and rodent enzymes (24, 25) is actually not conserved in this protein
family (see "Discussion"). In all sequences shown in Fig. 4, the
first aspartate is strictly conserved and is preceded most often by
four hydrophobic residues. The second aspartate is found in all
phosphotransferases and phosphatases acting on phosphomonoesters shown
in Fig. 4 but is replaced by a threonine in ATPases and by a tyrosine
in dehalogenases. The threonine found in position +4 with respect to
the phosphorylated aspartate is conserved in all enzymes except
-phosphoglucomutases and paranitrophenylphosphatases.
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Formation of a Phosphoenzyme with -Phosphoglucomutase--
The
results mentioned above imply that all phosphotransferases shown in
Fig. 4 should form, when incubated with their substrate, an
aspartyl-phosphoenzyme. Evidence for this formation has been provided
for ATPases, as well as for several of the phosphotransferases possessing the DXDXT motif, i.e. PMM,
PSP, and 2-phosphoglycolate phosphatase (26). It was of interest to
confirm this for
-phosphoglucomutase, in which the conserved
threonine is replaced by a valine.
-Phosphoglucomutase was partially
purified from L. lactis by chromatography on DEAE-Sepharose and was completely separated from
-phosphoglucomutase. Incubation of
the enzyme with [32P]glucose 6-phosphate in the presence
of glucose 1,6-bisphosphate resulted in the incorporation of
radioactivity into protein. SDS-PAGE analysis indicated that the
labeled peptide had a Mr of 25,000, in good
agreement with the molecular mass calculated from the sequence (24,210 Da). The phosphoenzyme bond was completely hydrolyzed after 10 min of
incubation in 1 M NaOH at 0 °C or in 0.2 M
NH2OH (pH 5.3) at 20 °C and about 40% hydrolyzed after
15 min of incubation at 37 °C in M HCl (results not
shown), in agreement with an acyl-phosphate bond (27).
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DISCUSSION |
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Identification of the Residue Phosphorylated in Phosphomannomutase 1-- The residue phosphorylated in PMM1 was hypothesized (13) to be an aspartate or a glutamate on the basis of the stability of the phosphoenzyme linkage (27). Its location in the sequence could be identified after reduction of phosphoaspartate to homoserine with radiolabeled borohydride, proteolytic digestion, and separation of the peptides by HPLC. The elution profile of the tryptic/chymotryptic peptides derived from the phosphorylated enzyme showed indeed an additional reduced peptide that could be unambiguously identified based on its molecular mass, the size of its fragments, and the known sequence of PMM1. The fragmentation results showed additionally that the phosphorylated residue was the first aspartate in the a DVDGT motif.
Two other reduced peptides could be detected in the digests of both control and phosphorylated enzymes. Both peptides could be identified by mass spectrometry, and the position of the reduced bond could be assigned to one of the first three residues of the peptide with m/z 1595.7 and to one of the first nine residues of the peptide with m/z 1469.5. In the case of the first peptide, this excludes reduction of a carboxyl-phosphate group, as the implicated sequence does not contain an aspartate or a glutamate residue. We speculate that the 14-amu loss is due to reduction of a peptide bond to a secondary amine due to a local effect of the amino acid sequence. The fact that both peptides were found with the control and the phosphorylated enzymes indicates that the underlying modifications are probably not related to the catalytic mechanism. Site-directed mutagenesis confirmed the importance of the two aspartate residues in the DVDGT motif of PMM; their rather conservative replacement by a glutamate or by an asparagine resulted in a complete loss of activity. The fact that the mutated proteins had conserved the same behavior upon chromatography indicated that the loss of activity was not due to a gross folding problem (28).Identification of the Phosphorylated Residue in L-3-Phosphoserine Phosphatase-- We could not detect the modified residue in PSP with the same methodology as applied for PMM, probably because the phosphorylation stoichiometry of this enzyme is about 10 times lower than that of the mutase (11, 13). The mutagenesis studies showed, however, that the first of the two aspartate in the conserved DXDST motif could not be replaced by glutamate or by asparagine without total loss of activity, whereas the second aspartate could be replaced by glutamate, although not by asparagine, with conservation of substantial activity. This result indicates that the carboxylic group of the first aspartate is much more position sensitive for its function than that of the second aspartate, which fits very well with the notion that it would participate in catalysis as a nucleophile, the second aspartate playing possibly a role as an acid-base catalyst (see below). These considerations, as well as the analogy with PMM, indicate that the first of the two aspartates is the phosphorylated residue.
A Family of Phosphotransferase with a DXDX(T/V)
Motif--
Sequence comparisons indicate that at least nine
phosphatases or phosphomutases contain a similar, totally conserved
DXDX(T/V) motif, which is located in the
amino-terminal region of the protein, or in the case of the
bifunctional enzyme, trehalose 6-phosphate synthase/trehalose
6-phosphate phosphatase, in the amino-terminal region of the trehalose
6-phosphatase domain. This motif is particularly striking in the case
of some enzymes like phosphoglycolate phosphatase, trehalose
6-phosphatase, PSP, and paranitrophenylphosphatase, in which only
8-14% of the residues are strictly conserved (not shown). For four of
these enzymes (phosphoglycolate phosphatase (26), PSP (11), PMM (13),
-phosphoglucomutase (this paper)), evidence for a phosphoenzyme
intermediate with the characteristics of a carboxyl-phosphate bond has
been provided. For one of them (PMM), the phosphorylated residue could
be definitely identified as the first of the two aspartates; for a
second one (PSP), site-directed mutagenesis is highly suggestive of
this (see above). We speculate that the same aspartate is
phosphorylated in the other members of this protein family. With at
least nine members, this new family of phosphotransferases seems to be
slightly larger than the bisphosphoglycerate mutase family, which
comprises two mutases and three phosphatases and in which the
phosphorylated residue is a histidine in a conserved RHG motif (29,
30).
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ACKNOWLEDGEMENTS |
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We thank H. G. Hers and M. Rider for helpful comments, E. de Hoffmann (Laboratoire de Spectrométrie de Masse, UCL) for giving access to mass spectrometry equipment, and G. Berghenouse for technical assistance.
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FOOTNOTES |
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* This work was supported by the Actions de Recherche Concertées, by the Belgian Federal Service for Scientific, Technical, and Cultural affairs, and by the Belgian Fonds National de la Recherche Scientifique.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.
§ Supported by the Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture.
¶ Aspirant of the Belgian Fonds National de la Recherche Scientifique.
To whom correspondence should be addressed: UCL 7539, avenue
Hippocrate 75, B-1200 Brussels, Belgium. Tel.: 32-2-7647564; Fax:
32-2-7647598; E-mail: vanschaftingen{at}bchm.ucl.ac.be.
1 The abbreviations used are: PSP, L-3-phosphoserine phosphatase; PMM, phosphomannomutase; Mes, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; amu, atomic mass units.
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REFERENCES |
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