©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Small Membrane-associated Sugar Phosphate Phosphatase That Is Allosterically Activated by HPr(Ser(P)) of the Phosphotransferase System in Lactococcus lactis(*)

Jing-Jing Ye , Milton H. Saier , Jr. (§)

From the (1)Department of Biology, University of California at San Diego, La Jolla, California 92093-0116

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the Gram-positive bacterium, Lactococcus lactis, nonmetabolizable cytoplasmic sugar phosphates, accumulated by the phosphoenolpyruvate:sugar phosphotransferase system, are rapidly dephosphorylated and expelled from the cell upon addition of glucose (inducer expulsion). Our recent studies have established that a metabolite-activated, ATP-dependent protein kinase that phosphorylates serine-46 in HPr of the phosphoenolpyruvate:sugar phosphotransferase system activates a sugar phosphate phosphatase, thus initiating the inducer expulsion process. A membrane-associated, HPr(Ser(P))-dependent phosphatase has been identified, solubilized from the membrane, separated from other cellular phosphatases, and purified to near homogeneity. It exhibits a low subunit molecular mass (10 kDa) and behaves on gel filtration columns like a monomeric enzyme. It has broad substrate specificity, optimal activity between pH 7.0 and 8.0, is dependent on a divalent cation for activity, and is not inhibited by fluoride. It is stimulated more than 10-fold by HPr(Ser(P)) or a mutant derivative of HPr, S46D HPr, in which the regulatory serine is changed to aspartate, which bears a permanently negative charge as does phosphate. Stimulation is due both to an increase in the maximal velocity (V) and a decrease in the Michaelis-Menten kinetic constant (K) for sugar phosphate. The enzyme exhibits a K for S46D HPr of 15 µM. Although the enzyme is thermally stable, activation by HPr(Ser(P)) is heat sensitive.


INTRODUCTION

The phenomenon of ``inducer expulsion'' is well established in Gram-negative bacteria such as Escherichia coli (Haguenauer and Képès, 1971; Winkler, 1971) and in Gram-positive bacteria such as Streptococcus pyogenes and Lactococcus lactis (Reizer and Panos, 1980; Thompson and Saier, 1981). Although the mechanism is still not understood in E. coli, recent work with Gram-positive bacteria has implicated the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS).()In these organisms, HPr is phosphorylated both by phosphoenolpyruvate and enzyme I of the PTS on histidyl residue 15 and by ATP and a metabolite-activated protein kinase on seryl residue 46 (Deutscher and Saier, 1983; Deutscher et al., 1986; Reizer et al., 1989). Kinetic analyses showed that inducer expulsion is a two step process: intracellular sugar phosphate is first hydrolyzed to free sugar and inorganic phosphate in the cytoplasm, and the free sugar is then expelled into the external medium (Reizer et al., 1983; Reizer and Saier, 1983; Sutrina et al., 1988). In early studies, HPr(Ser) phosphorylation and inducer expulsion appeared to be related since conditions that gave rise to inducer expulsion also promoted phosphorylation of HPr on serine (Thompson and Saier, 1981; Reizer et al., 1992).

Recent work employing vesicles of L. lactis has provided direct, compelling evidence for the involvement of HPr(Ser(P)) in (a) the inhibition of sugar uptake via the PTS, (b) the activation of a cytoplasmic sugar phosphate phosphatase, and (c) the consequent phenomena of inducer exclusion and expulsion (Saier, 1985; Ye et al., 1994a, 1994b). Activation of the sugar phosphate phosphatase by the HPr(Ser(P)) mutant analogue, S46D HPr (Reizer et al., 1989, 1992; Wittekind et al., 1989, 1990, 1992), has recently been demonstrated in permeabilized vesicles of L. lactis (Ye et al., 1994a, 1994b). Evidence for the involvement of HPr(Ser(P)) and S46D HPr in the regulation of non-PTS carbohydrate permeases in Lactobacillus brevis (Ye et al., 1994c, 1994d; Ye and Saier, 1995a, 1995b) and in catabolite repression in Bacillus subtilis (Deutscher et al., 1994, 1995; Hueck et al., 1994)()has recently been presented.

Previously published work did not lead to identification of the sugar phosphate phosphatase that is activated by HPr(Ser(P)) and S46D HPr. A sugar phosphate phosphatase was purified and characterized from L. lactis (Thompson and Chassy, 1983), but its involvement in inducer expulsion was not demonstrated. We therefore set out to identify the enzyme that is the target of HPr(Ser(P)) action. We were able to show that the phosphatase of Thompson and Chassy (here termed phosphatase I or Pase I) is not activated by S46D HPr. By contrast, a membrane-associated sugar phosphate phosphatase (here termed phosphatase II or Pase II) was found to be activated by S46D HPr. This enzyme could be solubilized from the membranes with the use of 8 M urea and 4% butanol and purified to near homogeneity. Its unusual properties and its functional interactions with HPr and various mutant derivatives of HPr are herein described.


EXPERIMENTAL PROCEDURES

Assay Conditions

Sugar phosphate phosphatase activity was routinely determined by a modification of the method described by Parvin and Smith(1969). Briefly, 10-25 µl of enzyme was added to a medium containing 50 mM MES (pH 7.0), 20 mM MgSO, and various concentrations (usually 10 or 20 mM) of one of several sugar phosphates. Assay solutions (50 µl, final volume) were incubated at 40 °C for 10-30 min. Isobutanol (1 ml) and 4.25% (w/v) ammonium molybdate in 0.43 M sulfuric acid (0.3 ml) were then added, and phosphate was extracted into the organic phase by vigorous mixing for 20 s. When the organic phase had cleared, 1 ml was removed and mixed by immediately vortexing with 1 µl of 4% (w/v) SnCl in concentrated HCl before measurement of the optical density at 760 nm.

[C]TMG-6-phosphate was prepared by using starved cells of L. lactis as described by Thompson and Saier(1981). [C]TMG-6-phosphate phosphatase activity was measured as follows. 1.5 mM [C]TMG-6-phosphate (specific activity, 0.5 mCi/mmol, purified as previously described (Ye et al., 1994a, 1994b)) was added to the same assay solution described above lacking a sugar phosphate substrate. The assay solution was then incubated for up to 2 h before measuring the proportion of free [C]TMG by ion exchange chromotography (5 0.5 cm, column size) as previously outlined (Kundig and Roseman, 1971). PTS enzyme I and HPr were assayed by complementation of mutant crude extracts as previously described (Mitchell et al., 1993). Protein concentration was determined by the procedure of Lowry et al.(1951).

Organism and Growth Conditions

L. lactis ML3 (12 liters for a typical enzyme preparation) was grown at 37 °C for 16 h in LB medium containing 25 mM galactose as previously described (Ye et al., 1994a, 1994b). A final yield of about 45 g (wet weight) of cells was usually obtained.

Purification of Sugar Phosphate Pase II

All purification procedures were conducted at 0-4 °C. The washed cell pellet (about 45 g) was resuspended to a volume of 60 ml in HEPES buffer (pH 7.3) containing 1 mM EDTA, 1 mM dithiothreitol, and 5 mM MnSO (HEDM buffer), as well as 0.1 mM phenylmethylsulfonyl fluoride. The cells were disrupted by three successive passages through a French pressure cell (11,000 pounds per inch (76 megapascal)). Cell debris was removed by centrifugation at 10,000 g for 20 min, and the membranes were collected by ultracentrifugation at 200,000 g for 90 min.

The membranes were resuspended in HEDM buffer and treated with 8 M urea and 4% 1-butanol at 0 °C for 30 min as previously described (Kundig and Roseman, 1971; Saier et al., 1977) before centrifugation at 200,000 g for 2 h. The supernatant was collected and dialyzed overnight against 11 liters of 20 mM Tris-HCl, pH 7.0.

The dialyzed, urea-butanol-treated supernatant (40 ml) was transferred (at a flow rate of 0.5 ml min) onto a DEAE-Sephacel anion-exchange column (3 20 cm) previously equilibrated with HEDM buffer. The proteins were eluted with a gradient of 0.1-0.4 M NaCl in HEDM buffer (see Fig. 1A).


Figure 1: Elution profiles of phosphatase I and phosphatase II from DEAE-Sephacel ion exchange (A) and of phosphatase II from Sephadex G-50 (B). Conditions were as outlined under ``Experimental Procedures.'' In A, elution was effected with a linear salt gradient of 0.1-0.4 M NaCl as indicated by the dashedline. Phosphatase II eluted at an estimated salt concentration of 0.33 M. In B, catalase (M = 220,000) eluted at the portion indicated by arrow1, and cytochrome c (M = 12,000) eluted at the portion indicated by arrow2.



Fractions from the DEAE-Sephacel column that contained Pase II activity were pooled and concentrated to 3 ml by lyophilization. After addition of 0.5 ml of glycerol, the enzyme was loaded onto a Sephadex G-50-50 column (1.2 32 cm) preequilibrated and eluted with HEDM buffer. Fractions containing sugar phosphate phosphatase activity were combined, analyzed for purity, and used for characterization of enzymic properties (see Fig. 1B). Although Pase II was of about the same size as HPr, it exhibited no detectable HPr activity in complementation assays (Mitchell et al., 1993). Reciprocally, all HPr and mutant HPr derivative preparations tested for activation of Pase II lacked detectable phosphatase activity. The Pase II preparation purified through gel filtration was used in all analyses reported except for those presented in .

Gel Electrophoretic Analysis

SDS-electrophoresis was performed with a PhastSystem Separation and Control Unit (Pharmacia, Uppsala, Sweden). 5 µl (from 0.1 to 5 µg of protein) of each sample was mixed with SDS sample buffer (60 mM Tris-HCl, pH 7.0, 2% SDS, 10% glycerol, 0.025% bromphenol blue) and boiled at 100 °C for 2 min before loading on a PhastGel (Gradient 8-25) for separation by SDS-polyacrylamide gel electrophoresis. The protein bands were visualized employing staining with Coomassie Blue R-250 by the procedure of Diezel et al.(1972).

Preincubation of Pase II at Various Temperatures

Aliquots of Pase II, purified through the gel filtration column, were incubated for 10 min in tubes submerged in a water bath at various temperatures ranging from 40 to 100 °C. They were then chilled to 0 °C in preparation for assay of the mannitol-1-P (20 mM) hydrolysis rate with or without 50 µM S46D HPr employing the standard assay conditions described above.

Reagents

Radiolabeled [C]TMG (58 mCi/mmol) was from DuPont-NEN. Bio-safe II scintillation mixture was purchased from Research Products International Corp. (Mt. Prospect, IL). Analytical gradient anion-exchange resin AG1-X2 was obtained from Bio-Rad. Sephadex G-50-50 was from Sigma, and DEAE-Sephacel anion-exchange resin was obtained from Pharmacia Biotech Inc. PhastGels (Gradient 8-25) for SDS-polyacrylamide gel electrophoresis separations were from Pharmacia Biotech AB (Uppsala, Sweden). Nonradioactive compounds, phosphorylated sugars, and other reagents were obtained from Sigma.


RESULTS

Solubilization and Purification of Pase II

Crude extracts of L. lactis exhibited low degrees of stimulation of mannitol-1-P and [C]TMG-phosphate phosphatase activities upon addition of S46D HPr (). When the membrane fraction was separated from the soluble protein fraction by high speed centrifugation (200,000 g for 2 h), the resuspended membranes exhibited activity that was enhanced by addition of S46D HPr to a greater extent than was observed for the crude extract. The soluble protein fraction exhibited more activity than did the membranes, but this activity exhibited almost no stimulation by S46D HPr (). As noted below, the soluble fraction was enriched for Pase I while the membranes were enriched for Pase II. Membranes were therefore used as the preferred source of Pase II.

The membranes were treated with 8 M urea and 4% butanol essentially as previously described (Kundig and Roseman, 1971; Saier et al., 1977) to release Pase II. After centrifugation to remove the membranes, and after removal of the butanol and urea by dialysis, the enzyme was purified by ion exchange chromatography and gel filtration as described under ``Experimental Procedures.'' An estimate of the -fold purification was not possible because of the presence of both Pase I and Pase II and possibly other sugar phosphate phosphatases. However, an approximately 50-fold increase in specific activity was observed during purification of the activity released from the membranes by butanol-urea extraction.

Fig. 1presents the elution profiles of activity from the DEAE-Sephacel I column (Fig. 1A) and the Sephadex G-50-50 gel filtration column (Fig. 1B). Pase I eluted from the ion exchange column at a salt concentration of about 0.2 M while Pase II eluted at about 0.33 M NaCl. The two activity peaks were clearly separated. While Pase I activity was not detectably stimulated by S46D HPr, Pase II activity was stimulated 5-10-fold ().

Gel filtration (Fig. 1B) of the concentrated Pase II off of the DEAE-Sephacel column yielded a single major peak of activity, which eluted shortly after cytochrome c (M 12,000, see Fig. 1B). Its size was estimated at 9,000-10,000 Da. Gel electrophoresis revealed a single band that ran just ahead of HPr using the Coomassie Blue protein stain (data not shown). Pase II gave the following N-terminal sequence: APLKGRF. Only a single predominant amino acid (>80%) was present at each position. The amounts of these amino acids, relative to each other, were about the same, suggesting that they derived from a single protein. This enzyme was used for all remaining experiments reported below.

Substrate Specificity of Pase II

presents data revealing the substrate specificity of the enzyme and its -fold stimulation by S46D HPr with each of the potential substrates tested. Almost all sugar phosphates tested were hydrolyzed, although to varying degrees, and the -fold stimulation by S46D HPr varied between 5- and 10-fold. In no case did S46A HPr stimulate the activity of Pase II. Fructose 1,6-diphosphate and p-nitrophenyl phosphate were poor substrates, and the low activity observed with these substrates was not appreciably stimulated by S46D HPr.

Dependence of Pase II Activity on Divalent Cations

Using mannitol-1-P as the substrate, Pase II activity was found to be dependent on divalent cations. Regardless of concentration, Mg appeared to be most effective. Mg stimulated half-maximally at a concentration of about 1 mM, and high concentrations were not inhibitory (data not shown). The stimulatory effect of Mg was independent of S46D HPr. Other divalent cations (Mn, Co, Fe, and Zn) stimulated to lesser degrees with decreasing efficacies in the order mentioned. Fluoride, which inhibited Pase I almost quantitatively under our standard assay condition (see also Thompson and Chassy, 1983), had no effect on the activity of Pase II when present in 2-fold excess over the Mg concentration.

pH Dependence of Pase II

The pH curves with and without S46D HPr are shown in Fig. 2. In both cases, the pH optimum was about 7 to 8. Stimulation by S46D HPr was maximal within this pH range.


Figure 2: pH activity curve for purified phosphatase II. Buffers used at 50 mM were as follows: pH 3-5, sodium acetate; pH 6-8, MES buffer adjusted to the appropriate pH with HCl; pH 9-10, sodium borate. Other conditions were as outlined under ``Experimental Procedures'' with 20 mM mannitol-1-P as the substrate.



Kinetic Characteristics of S46D HPr-stimulated Pase II Activity

In the absence of S46D HPr, Pase II exhibited low affinity for its sugar substrates. For example, the apparent K for mannitol-1-P was 50 mM. When 35 µM S46D HPr was added, the K decreased to about 7 mM, and the V increased about 50% (Fig. 3). No apparent cooperativity (sigmoidicity of the vversusS curve) was observed either in the presence or the absence of S46D HPr.


Figure 3: Lineweaver-Burk, double reciprocal plot of the mannitol-1-P hydrolysis rate as a function of the sugar phosphate concentration. Conditions other than substrate concentration were as outlined under ``Experimental Procedures'' with 50 mM MES buffer, pH 7.0. Phosphatase II, purified through gel filtration, was used in the assay. The calculated K and V values at 40 °C were as follows: without S46D HPr, 50 mM and 2.0 µmol/mg protein/min, respectively, and with S46D HPr at 35 µM, 8 mM and 3.8 µmol/mg protein/min, respectively.



The dependence of the activity on S46D HPr is shown in Fig. 4. Under the assay conditions used, 50 µM S46D HPr gave maximal activity with half-maximal activation occurring at about 13 µM. The curve was hyperbolic, consistent with a lack of cooperativity for substrate binding. In agreement with these results, chemical cross-linking experiments with disuccinimidyl tartarate conducted as described by Belogrudov et al.(1995) revealed that Pase II alone or S46D alone each gave only one band on SDS-polyacrylamide gel electrophoresis at 9-10 kDa, but when these two proteins were mixed prior to the addition of disuccinimidyl tartarate, an additional band of about 18-20 kDa was observed. A 1:1 stoichiometry of binding is therefore suggested.


Figure 4: Activation of phosphatase II as a function of S46D HPr concentration with 20 mM mannitol-1-P as the substrate in the presence of 50 mM MES buffer, pH 7.0, and 20 mM MgCl under standard assay conditions. The K for S46D HPr was calculated to be 13 µM.



Stimulatory Effects of Various HPr Derivatives on Pase II Activity

I presents the effects of HPr, HPr(Ser(P)), and various mutant derivatives of HPr on the activity of the purified Pase II. When used at a concentration of 15 µM, HPr(Ser(P)) was substantially more stimulatory than was S46D HPr. When used at near saturating concentrations (50 µM), S46D HPr and the double mutant, H15E,S46D HPr, were equally stimulatory. All other derivatives of HPr tested were essentially without effect (I).

Thermal Stability of Pase II and Thermal Inactivation of the S46D Stimulatory Effect

Fig. 5shows the effects of a short preincubation of Pase II at temperatures ranging from 40 to 100 °C on the activities of the purified enzyme. Basal Pase II activity using mannitol-1-P as substrate was unaffected by thermal exposure, even at a preincubation temperature of 100 °C. By contact, the stimulatory effect of S46D HPr was gradually lost to increasing degrees when the enzyme was exposed to temperatures ranging from 50 to 100 °C. The enzyme preincubated at 100 °C exhibited little stimulation. The results suggest that while the active site of the enzyme is heat stable, the S46D HPr interaction site is less stable.


Figure 5: Effects of preincubation at various temperatures on Pase II activity with and without S46D HPr. The purified enzyme was heated 10 min at the temperature indicated, chilled at 0 °C, and assayed with or without S46D HPr at a concentration of 50 µM. The assays were performed at 40 °C with 20 mM mannitol-1-P as substrate as outlined under ``Experimental Procedures.''




DISCUSSION

Inducer expulsion in Gram-positive bacteria possessing a PTS is mediated by a metabolite-activated, ATP-dependent protein kinase. A cytoplasmic sugar phosphate phosphatase has been implicated in the process (See Introduction). Prior to this report, nothing was known about the enzyme that catalyzed this reaction. Using S46D HPr stimulation of sugar phosphate phosphatase activity as an assay, we have purified a phosphatase (termed Pase II) from L. lactis extracts that exhibits unusual properties as follows. 1) It has an exceptionally small catalytic subunit, possessing a molecular mass of less than 10,000 Da, based on both gel filtration of the native enzyme and SDS-gel electrophoresis of the denatured enzyme. A small size had been suggested early in our studies by the observation that the enzyme passed through dialysis tubing at a rate comparable to that of HPr.()2) While Pase II elutes from DEAE Sephacel ion exchange columns at a high (0.33 M) NaCl concentration, Pase I elutes at a much lower salt concentration (0.2 M), suggesting that Pase II may be a much more acidic protein than is Pase I. 3) The activity of Pase II, like that of Pase I, is stimulated by various divalent metal ions, with Mg being preferred. However, unlike most phosphatases, including Pase I (Thompson and Chassy, 1983), fluoride is apparently not inhibitory under the assay conditions used. 4) In contrast to Pase I, which has a low pH optimum (Thompson and Chassy, 1983), Pase II exhibits a high pH optimum. 5) Pase II seems to be predominantly peripherally associated with the particulate fraction while Pase I is predominantly soluble in crude extracts. The former enzyme can be quantitatively solubilized by treatment with 8 M urea in the presence of 4% butanol according to the procedure of Kundig and Roseman(1971). This fact suggests that the enzyme is not an integral constituent of the membrane. 6) In contrast to Pase I, which is not affected by HPr and its derivatives, Pase II is specifically activated by HPr(Ser(P)), S46D HPr, and H15E,S46D HPr. Non-phosphorylated wild-type HPr and other mutant derivatives of HPr did not activate Pase II. The binding stoichiometry is apparently 1:1. These facts suggest that Pase II directly binds HPr(Ser(P)) to initiate inducer expulsion in lactococci. 7) Physiological concentrations of S46D HPr both lower the K for sugar phosphate and increase the V for sugar phosphate hydrolysis, thereby accounting for stimulation of the activity of Pase II. It is interesting to note that while Pase II is activated by H15E,S46D HPr as well as S46D HPr, only the latter HPr derivative could bind to the CcpA protein, a mediator of catabolite repression in B. subtilis(8) . Finally, while Pase II activity appears to be heat stable, its activation by S46D HPr is heat labile. This last observation may suggest that the interaction of Pase II with S46D HPr is dependent on a conformation of the enzyme that is not required for catalysis.

The results summarized in this paper serve to characterize the phosphatase that is most probably responsible for the inducer expulsion phenomenon in L. lactis and possibly in other Gram-positive bacteria as well. Its small size suggests that it will prove ideal material for structural studies employing both multidimensional NMR and x-ray crystallography. Since high resolution three-dimensional structures of HPr are already available (Wittekind et al., 1989, 1990, 1992; Herzberg et al., 1992), the Pase II-HPr(Ser(P)) interaction should be molecularly definable (Chen et al., 1993).

  
Table: Stimulation of phosphatase activity in a crude extract of L. lactis and in various partially purified fractions by S46D HPr

Assay conditions were as reported under ``Experimental Procedures'' with mannitol-1-P (20 mM) as the substrate. Values of three experiments were averaged and are reported ± S.D.


  
Table: Substrate specificity of S46D HPr-stimulated sugar phosphate phosphatase II from L. Lactis

Phosphatase II (15 µl of 0.2 mg/ml of the purified enzyme) was incubated in an assay solution containing 20 mM Mg and 50 mM MES buffer (pH 7.0) for 15 min at 40 °C as detailed under ``Experimental Procedures.'' The substrate concentration was 20 mM. When TMG-6-phosphate was the substrate, 0.2 mM [C]TMG-6-phosphate (specific activity, 0.5 mCi/mmol) was present in the assay solution. HPr derivatives were used at a concentration of 100 µM. Values of three experiments were averaged and are reported ± S.D.


  
Table: Stimulation of purified phosphatase II by various HPr derivatives

Assays were performed as indicated under ``Experimental Procedures.'' 20 mM mannitol-1-P was present in all assays as the substrate. Values of three experiments were averaged and are reported ± S.D.



FOOTNOTES

*
This work was supported by Public Health Service Grants 5RO1AI21702 and 2RO1AI14176 and National Institutes of Health Postdoctoral Fellowship 1F33GM16907 (to J. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 619-534-4084; Fax: 619-534-7108; E-mail: msaier@ucsd.edu.

The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; HPr, the heat-stable phosphocarrier protein of the PTS; Pase I, phosphatase I; Pase II, phosphatase II; [C]TMG, -D-[methyl-C]thiogalactopyranoside; MES, 2-(N-morpholino)ethanesulfonic acid.

J. Reizer, V. Bergstedt, E. Kuster, V. Charrier, M. H. Saier, Jr., W. Hillen, M. Steinmetz, and J. Deutscher, submitted for publication.

J.-J. Ye and M. H. Saier, Jr., unpublished observations.


ACKNOWLEDGEMENTS

We thank Mary Beth Hiller and Lisa Preble for assistance in the preparation of this manuscript.


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