The phosphatase C(X)5R motif is required for catalytic activity of the Saccharomyces cerevisiae Acr2p arsenate reductase.

Acr2p detoxifies arsenate by reduction to arsenite in Saccharomyces cerevisiae. This reductase has been shown to require glutathione and glutaredoxin, suggesting that thiol chemistry might be involved in the reaction mechanism. Acr2p has a HC(X)(5)R motif, the signature sequence of the phosphate binding loop of the dual-specific and protein-tyrosine phosphatase family. In Acr2p these are residues His-75, Cys-76, and Arg-82, respectively. Acr2p has another sequence, (118)HCR, that is absent in phosphatases. Acr2p also has a third cysteine residue at position 106. Each of these cysteine residues was changed individually to serine residues, whereas the histidine and arginine residues were altered to alanines. Cells of Escherichia coli heterologously expressing the majority of the mutant ACR2 genes retained wild type resistance to arsenate, and the purified altered Acr2p proteins exhibited normal enzymatic properties. In contrast, cells expressing either the C76S or R82A mutations lost resistance to arsenate, and the purified proteins were inactive. These results suggest that Acr2p utilizes a phosphatase-like Cys(X)(5)Arg motif as the catalytic center to reduce arsenate to arsenite.     

An arsenate reductase from Synechocystis sp. strain PCC 6803 exhibits a novel combination of catalytic characteristics

The deduced protein product of open reading frame slr0946 from Synechocystis sp. strain PCC 6803, SynArsC, contains the conserved sequence features of the enzyme superfamily that includes the low-molecular-weight protein-tyrosine phosphatases and the Staphylococcus aureus pI258 ArsC arsenate reductase. The recombinant protein product of slr0946, rSynArsC, exhibited vigorous arsenate reductase activity (V(max) = 3.1 micro mol/min. mg), as well as weak phosphatase activity toward p-nitrophenyl phosphate (V(max) = 0.08 micro mol/min. mg) indicative of its phosphohydrolytic ancestry. pI258 ArsC from S. aureus is the prototype of one of three distinct families of detoxifying arsenate reductases. The prototypes of the others are Acr2p from Saccharomyces cerevisiae and R773 ArsC from Escherichia coli. All three have converged upon catalytic mechanisms involving an arsenocysteine intermediate. While SynArsC is homologous to pI258 ArsC, its catalytic mechanism exhibited a unique combination of features. rSynArsC employed glutathione and glutaredoxin as the source of reducing equivalents, like Acr2p and R773 ArsC, rather than thioredoxin, as does the S. aureus enzyme. As postulated for Acr2p and R773 ArsC, rSynArsC formed a covalent complex with glutathione in an arsenate-dependent manner. rSynArsC contains three essential cysteine residues like pI258 ArsC, whereas the yeast and E. coli enzymes require only one cysteine for catalysis. As in the S. aureus enzyme, these "extra" cysteines apparently shuttle a disulfide bond to the enzyme's surface to render it accessible for reduction. SynArsC and pI258 ArsC thus appear to represent alternative branches in the evolution of their shared phosphohydrolytic ancestor into an agent of arsenic detoxification.     

A novel arsenate reductase from the arsenic hyperaccumulating fern Pteris vittata

Pteris vittata sporophytes hyperaccumulate arsenic to 1% to 2% of their dry weight. Like the sporophyte, the gametophyte was found to reduce arsenate [As(V)] to arsenite [As(III)] and store arsenic as free As(III). Here, we report the isolation of an arsenate reductase gene (PvACR2) from gametophytes that can suppress the arsenate sensitivity and arsenic hyperaccumulation phenotypes of yeast (Saccharomyces cerevisiae) lacking the arsenate reductase gene ScACR2. Recombinant PvACR2 protein has in vitro arsenate reductase activity similar to ScACR2. While PvACR2 and ScACR2 have sequence similarities to the CDC25 protein tyrosine phosphatases, they lack phosphatase activity. In contrast, Arath;CDC25, an Arabidopsis (Arabidopsis thaliana) homolog of PvACR2 was found to have both arsenate reductase and phosphatase activities. To our knowledge, PvACR2 is the first reported plant arsenate reductase that lacks phosphatase activity. CDC25 protein tyrosine phosphatases and arsenate reductases have a conserved HCX5R motif that defines the active site. PvACR2 is unique in that the arginine of this motif, previously shown to be essential for phosphatase and reductase activity, is replaced with a serine. Steady-state levels of PvACR2 expression in gametophytes were found to be similar in the absence and presence of arsenate, while total arsenate reductase activity in P. vittata gametophytes was found to be constitutive and unaffected by arsenate, consistent with other known metal hyperaccumulation mechanisms in plants. The unusual active site of PvACR2 and the arsenate reductase activities of cell-free extracts correlate with the ability of P. vittata to hyperaccumulate arsenite, suggesting that PvACR2 may play an important role in this process.     

Purification and characterization of ACR2p, the Saccharomyces cerevisiae arsenate reductase

In Saccharomyces cerevisiae, expression of the ACR2 and ACR3 genes confers arsenical resistance. Acr2p is the first identified eukaryotic arsenate reductase. It reduces arsenate to arsenite, which is then extruded from cells by Acr3p. In this study, we demonstrate that ACR2 complemented the arsenate-sensitive phenotype of an arsC deletion in Escherichia coli. ACR2 was cloned into a bacterial expression vector and expressed in E. coli as a C-terminally histidine-tagged protein that was purified by sequential metal chelate affinity and gel filtration chromatography. Acr2p purified as a homodimer of 34 kDa. The purified protein was shown to catalyze the reduction of arsenate to arsenite. Enzymatic activity as a function of arsenate concentration exhibited an apparent positive cooperativity with an apparent Hill coefficient of 2.7. Activity required GSH and glutaredoxin as the source of reducing equivalents. Thioredoxin was unable to support arsenate reduction. However, glutaredoxins from both S. cerevisiae and E. coli were able to serve as reductants. Analysis of grx mutants lacking one or both cysteine residues in the Cys-Pro-Tyr-Cys active site demonstrated that only the N-terminal cysteine residue is essential for arsenate reductase activity. This suggests that during the catalytic cycle, Acr2p forms a mixed disulfide with GSH before being reduced by glutaredoxin to regenerate the active Acr2p reductase.     

Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator

Root extracts from the arsenic (As) hyperaccumulating Chinese brake fern (Pteris vittata) were shown to be able to reduce arsenate to arsenite. An arsenate reductase (AR) in the fern showed a reaction mechanism similar to the previously reported Acr2p, an AR from yeast (Saccharomyces cerevisiae), using glutathione as the electron donor. Substrate specificity as well as sensitivity toward inhibitors for the fern AR (phosphate as a competitive inhibitor, arsenite as a noncompetitive inhibitor) was also similar to Acr2p. Kinetic analysis showed that the fern AR had a Michaelis constant value of 2.33 mM for arsenate, 15-fold lower than the purified Acr2p. The AR-specific activity of the fern roots treated with 2 mM arsenate for 9 d was at least 7 times higher than those of roots and shoots of plant species that are known not to tolerate arsenate. A T-DNA knockout mutant of Arabidopsis (Arabidopsis thaliana) with disruption in the putative Acr2 gene had no AR activity. We could not detect AR activity in shoots of the fern. These results indicate that (1) arsenite, the previously reported main storage form of As in the fern fronds, may come mainly from the reduction of arsenate in roots; and (2) AR plays an important role in the detoxification of As in the As hyperaccumulating fern.     

Structural characterization of the As/Sb reductase LmACR2 from Leishmania major

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 Å resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.     

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.     

Modulation of rat liver hydroxymethylglutaryl-CoA reductase by protein phosphatases: purification of nonspecific hydroxymethylglutaryl-CoA reductase phosphatases

Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature.     

A CDC25 homologue from rice functions as an arsenate reductase

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.     

G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells

Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.     

Distinction between substrate- and enzyme-directed effects of modifiers of rabbit liver phosphorylase a phosphatases

The natural substrate (phosphorylase a) and two alternative ones (phosphorylated histone and a tetradecapeptide consisting of residues 5-18 of rabbit skeletal muscle phosphorylase a) were used to distinguish the modes of action of some physiologically important effectors of four different molecular forms of rabbit liver phosphorlase a phosphatases. In general, glucose, caffeine, AMP, ADP, Pi, and glucose-1-P showed substrate-directed effects for the holophosphatase forms, since they usually did not affect the activity on histone phosphate and, with one slight exception (Pi), never affected the activity on the tetradecapeptide phosphate. ADP, Pi, and glucose-1-P did affect directly the relative mass (Mr) 35,000 phosphatase, in addition to an inhibition mediated via phosphorylase a. ATP exerted both substrate- and enzyme-directed effects for the Mr 35,000 phosphatase and phosphatases 1 and 2A2, but only a substrate-directed effect for phosphatase 2A1, suggesting that the gamma-subunit of the type 2 phosphatases may prevent ATP binding to the phosphatase. Mg2+ showed substrate-directed effects for phosphatases 1, 2A1, and 2A2, and an additional enzyme-directed effect for the Mr 35,000 phosphatase form. Furthermore, Mg2+ could not abolish ATP inhibition of the tetradecapeptide phosphatase activity, but significantly overcame ATP inhibition of the phosphorylase a phosphatase activity, thus suggesting that its ability to reverse the ATP effect is by a substrate-directed mechanism. The substrate-directed effects seen for the different ligands on the different phosphatase forms strongly indicate the significance of this form of control in the regulation of phosphorylase a phosphatase activities and may serve to narrow the otherwise broad substrate specificities of the major phosphorylase a phosphatase activities in mammalian tissues: phosphatases 1 and 2A.     

Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities

Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.     

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2)

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.     

In vitro dephosphorylation inhibits the activity of soybean lysine-ketoglutarate reductase in a lysine-regulated manner

In plant seeds, the essential amino acid lysine auto-regulates its own level by modulating the activity of its catabolic enzyme lysine-ketoglutarate reductase via an intracellular signaling cascade, mediated by Ca²⁺ and protein phosphorylation/dephosphorylation. In the present report, it has been further tested whether the activity of soybean lysine-ketoglutarate reductase, as well as that of saccharopine dehydrogenase, the second enzyme in the pathway of lysine catabolism, are modulated by direct phosphorylation of the bifunctional polypeptide containing both of these linked activities. Incubation of purified lysine-ketoglutarate reductase/ saccharopine dehydrogenase with casein kinase II resulted in a significant phosphorylation of the bifunctional enzyme. Moreover, in vitro dephosphorylation of the bifunctional polypeptide with alkaline phosphatase significantly inhibited the activity of lysine-ketoglutarate reductase, but not of its linked enzyme saccharopine dehydrogenase. The inhibitory effect of alkaline phosphatase on lysine-ketoglutarate reductase activity was dramatically stimulated by binding of lysine to the enzyme. Our results suggest that in plant seeds, active lysine-ketoglutarate reductase is a phospho-protein, and that its activity is modulated by opposing actions of protein kinases and phosphatases. Moreover, this modulation is subject to a compound regulation by lysine.     

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).     

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

Molecular cloning and characterization of a novel dual-specificity phosphatase18 gene from human fetal brain

Dual-specificity protein phosphatases (DSPs), a new family of protein tyrosine phosphatases (PTPs), are characterized by the ability to dephosphorylate both phospho-tyrosyl and phospho-seryl/threonyl residues. It has been known that most of the enzymes play important roles in the regulation of mitogenic signal transduction and control the cell cycle in response to extracellular stimuli. In this study, a novel human DSP gene named Dual-specificity Phosphatase18 (DUSP18) was isolated by large-scale sequencing analysis of a human fetal brain cDNA library. DUSP18 is localized at Chromosome 22 q12.1. Its cDNA is 2450 base pairs in length, encoding a 188-amino acid polypeptide in which a DSP motif but not a CH2 domain is included. RT-PCR revealed that the DUSP18 was widely expressed in different tissues. GST-DUSP18 fusion protein showed distinctive phosphatase activity toward p-nitrophenyl phosphate (pNPP), as well as oligopeptides containing pThr and pTyr, indicating that DUSP18 is a protein phosphatase with dual substrate specificity. The optimal condition for the reaction was pH 6.0 and 55 degrees C. Addition of Mn(2+) ions was able to enhance the enzyme activity while the activity was strongly inhibited by iodoaretic acid. Mutations in selected sites showed the importance of Asp-73, Cys-104, Arg-110 and Ser-111 in phosphatase activity of DUSP18.     

The Saccharomyces cerevisiae PHM8 gene encodes a soluble magnesium-dependent lysophosphatidic acid phosphatase

Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.     

Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator

The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.