©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Properties of Periplasmic 3`:5`-Cyclic Nucleotide Phosphodiesterase
A NOVEL ZINC-CONTAINING ENZYME FROM THE MARINE SYMBIOTIC BACTERIUM VIBRIO FISCHERI(*)

(Received for publication, January 23, 1995; and in revised form, March 28, 1995)

Sean M. Callahan (1), Neal W. Cornell (2), Paul V. Dunlap (1)(§)

From the  (1)Biology Department, Woods Hole Oceanographic Institution and the (2)Marine Biological Laboratory, Woods Hole, Massachusetts 02543

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 3`:5`-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm, allows this symbiotic bacterium to utilize extracellular 3`:5`-cyclic nucleotides (e.g. cAMP) as sole sources of carbon and energy, nitrogen, and phosphorus for growth. The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and Cibacron Blue 3GA-agarose. The active enzyme consists of a single polypeptide with a mass of 34 kDa. At 25 °C, it has a pH optimum of 8.25, a K for cAMP of 73 µm, and a V of 3700 µmol of cAMP hydrolyzed/min/mg protein (turnover number of 1.24 10/min). The specific activity of the V. fischeri enzyme is approximately 20-fold greater than that of any previously characterized CNP when comparisons of activity are made at the same assay temperature. Activity increases with temperature up to 60 °C. The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can restore activity of the apoenzyme to varying degrees. The exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacterium to scavenge 3`:5`-cyclic nucleotides in seawater and that the enzyme plays a role in cAMP-mediated host-symbiont interactions.


INTRODUCTION

3`:5`-Cyclic nucleotide phosphodiesterase (EC 3.1.4.17; CNP)()catalyzes the hydrolysis of 3`:5`-cyclic nucleotides (e.g. cAMP and cGMP) to their corresponding 5`-nucleoside monophosphates. The enzyme typically is located in the cytoplasm of cells. In eukaryotic organisms, several isozymes function in a variety of signal-mediated processes by modulating cytoplasmic levels of cAMP and cGMP(1) . Many prokaryotes also produce a cytoplasmic CNP. In bacteria, cAMP regulates gene transcription via interaction with a cAMP receptor protein. However, rather than CNP-mediated hydrolysis of cAMP or excretion from the cytoplasm, regulation of the synthesis of cAMP is thought to control cellular levels in bacteria(2, 3) . A CNP with atypical locations in the cell has been described from the cellular slime mold Dictyostelium discoideum. That enzyme is unique in occurring as both extracellular and cell membrane-associated forms, which catalyze the hydrolysis of extracellular cAMP involved in morphogenetic and aggregational signaling during plasmodium formation(4, 5) .

We recently described a second extra-cytoplasmic CNP. The enzyme occurs in the marine bacterium Vibrio fischeri, which establishes a luminescent mutualism with certain marine animals. Its periplasmic location and high activity confer on V. fischeri the novel ability to utilize extracellular 3`:5`-cyclic nucleotides as sources of carbon and energy, nitrogen, and phosphorus for growth(6) . The gene, cpdP, for this enzyme is the only bacterial CNP gene that has been cloned. The deduced amino acid sequence exhibits 34% identity with the extracellular CNP of D. discoideum and 30% identity with the low affinity CNP (PDE1) of the yeast Saccharomyces cerevisiae(7) . The V. fischeri enzyme is specific for 3`:5`-cyclic nucleotides (6) and, therefore, differs from previously described bacterial periplasmic phosphatases such as 2`:3`-cyclic phosphodiesterase:3`-nucleotidase (EC 3.1.4.16) and 5`-nucleotidase (EC 3.1.3.5).

The V. fischeri enzyme has been proposed to play a central role in the luminescent (light organ) symbiosis of V. fischeri with sepiolid squids and monocentrid fish(6) . Since periplasmic CNP activity is rare in bacteria(6) , the enzyme may contribute to the specificity of the symbiosis by permitting V. fischeri cells to utilize putative host-released cAMP as a nutrient. Alternatively, the enzyme may function to degrade 3`:5`-cyclic nucleotides released from organisms into the marine environment(6, 7) .

In this report, we describe the purification and biochemical properties of the V. fischeri periplasmic CNP. A detailed understanding of the enzyme may lead to insights into the physiological and ecological roles of bacterial CNPs and the possibility of cAMP-mediated symbiotic interactions between V. fischeri and its hosts.


MATERIALS AND METHODS

Reagents

Myokinase (360 units/mg), pyruvate kinase (200 units/mg), lactate dehydrogenase (550 units/mg), and crystalline bovine serum albumin were purchased from Boehringer Mannheim. 5`-Nucleotidase (Crotalus adamanteus venom), 5`-ATP (disodium salt), NADH (disodium salt), 3`:5`-cyclic nucleotides, Trizma grade tris, phosphenolpyruvate (tri-cyclohexylammonium salt), Cibacron Blue-agarose 3GA, DEAE-Sephacel, and Sephacryl HR200 were obtained from Sigma, and Coomassie R-250 was from Fisher Chemical Co. (Pittsburgh, PA).

Bacterial Strains and Culture Conditions

V. fischeri strain MJ-1, from the light organ of the monocentrid fish Monocentris japonicus(8) , was maintained on LBS agar (6) at room temperature. Liquid cultures were grown at 26 °C with aeration (150 revolutions/min) in minimal medium containing 300 mM NaCl, 10 mM KCl, 50 mM MgSO, 10 mM CaCl, 15 mM NHCl, 0.3 mM -glycerophosphate, 20 mg/liter ferric ammonium citrate, 10 mM glucose, and 100 mM HEPES, pH 7.5. Cultures for enzyme purification were initiated with a 1% inoculum that had been grown to mid-exponential phase in minimal medium. Cells were harvested by centrifugation approximately 3 h after cultures attained stationary phase, at which point they produced a high level of luminescence.

Cell Fractionation

Cells were separated into outer membrane, inner membrane, and cytoplasmic/periplasmic (i.e. soluble) fractions as described for Vibrio cholerae(9) .

CNP Activity Assays

Three assays were used for measurement of CNP activity. In assay 1, the standard method used in this study, rates of hydrolysis of cAMP to AMP were measured by a modification of a coupled assay with adenylate kinase, pyruvate kinase, and lactate dehydrogenase(10) . Specifically, identical reference and reaction mixtures consisted of 2 mM cAMP (except where otherwise indicated), 0.6 mM ATP, 0.17 mM NADH, 0.5 mM phosphoenolpyruvate, 4 units of adenylate kinase, 4 units of pyruvate kinase, 10 units of lactate dehydrogenase, 20 mM KCl, 5 mM MgCl, and 100 mM Tris-HCl, pH 8.25, in a volume of 2 ml. The reaction was initiated by addition of samples containing CNP, and the production of NAD from NADH was monitored spectrophotometrically as the change in absorbance at 340 nm. Consistent with the coupled nature of the assay, the reaction exhibits a lag that is inversely proportional to the amount of CNP added. Following the lag, the reaction rate is linear up to a A of 0.8. CNP activity was calculated from the linear portion of the reaction using the extinction coefficient of 6.22/mM/cm (11) taking into account that 2 mol of NAD are produced per mole of cAMP hydrolyzed. One unit of CNP activity is defined as the activity that catalyzes the hydrolysis of 1 µmol of cAMP in 1 min.

Control experiments demonstrated that the reaction rates obtained with assay 1 were linearly dependent on the amount of CNP in the reaction mixture over a 4-fold range (0.04-0.164 µmol of NADH oxidized/min), indicating that the observed CNP activity was not limited by any of the rate indicator components. This finding was confirmed at each temperature used in the determinations of K and V. Furthermore, since adenosine was found to be a competitive inhibitor of CNP (see ``Results''), control experiments were conducted and verified that ATP (included in the assay at 0.6 mM as an adenylate kinase substrate) does not inhibit CNP activity.

Assay 2, a two-stage version of assay 1, was developed to examine the effects of pH, temperature, and metal ions on CNP activity without interference of these effects on the activities of the coupling enzymes. In the first stage, samples containing CNP were added to reaction mixtures containing 100 mM Tris-HCl, pH 8.25, and 6 mM cAMP in a volume of 1.0 ml; the reactions were initiated by the addition of the CNP and were terminated at various times by the addition of 53 µl of 60% (w/v) perchloric acid, which irreversibly inactivated the CNP. This mixture was placed on ice, and 142 µl of 3 M KOH was then added to raise the pH to approximately 6 and to precipitate the perchlorate anion. In the second stage, 20 µl of the perchlorate/KOH-treated reaction mixture was added to a mixture containing the components of Assay 1 except for the CNP and cAMP. The total A was used to calculate the amount of cAMP hydrolyzed to AMP during the first-stage reaction. Rates were linear with respect to the time of the first reaction. Rate measurements made with assay 2 agreed within 10% of those made with assay 1.

Assay 3, used for substrates other than cAMP, was a modification of a previously described method (6, 12) with the following additional changes: 100 mM Tris-HCl, pH 8.25 at 25 °C as the reaction buffer, without MnCl which was not required for V. fischeri CNP activity, and with a 5`-nucleotidase reaction time of 30 min. Unless otherwise indicated, assays 1-3 were conducted at 25 °C. Protein concentrations were determined by the method of Bradford (13) using bovine serum albumin as the standard.

Purification

Periplasmic proteins were released from intact V. fischeri cells by a chloroform shock method(14) . Cells from two 1-liter cultures were collected by centrifugation (10,000 g, 10 min) and resuspended in 100 ml of 50 mM Tris-HCl, pH 8.0 (buffer 1) at 4 °C. 20 ml of chloroform was added to the cell suspension, which was inverted once to mix and then held at room temperature for 10 min. An additional 200 ml of buffer 1 was added, after which cells and chloroform were pelleted by centrifugation (16,000 g, 20 min, 4 °C). Buffer 1 was added to the supernatant (periplasmic extract) to adjust its protein concentration to 1 mg/ml. All subsequent steps of the purification were conducted at 4 °C.

The periplasmic extract was fractionated with ammonium sulfate, and the protein that precipitated between 40 and 70% of saturation was collected by centrifugation (16,000 g, 10 min) and dissolved in 10 ml of 10 mM imidazole buffer, pH 7.5 (buffer 2). The protein solution was then dialyzed for 18 h against three 1-liter volumes of buffer 2, changed at 0, 6, and 12 h, to remove the residual ammonium sulfate.

The dialyzed ammonium sulfate fraction was applied to 2 ml of packed DEAE-Sephacel equilibrated with buffer 2 in a 10-ml Poly-Prep column (Bio-Rad). 10 ml of 50 mM NaCl in buffer 2 was run through the column and discarded. CNP was then eluted from the column with 10 ml of 100 mM NaCl in buffer 2. The entire eluate was immediately applied to 1 ml of Cibacron Blue-agarose equilibrated with 100 mM MgSO in a second 10-ml Poly-Prep column. An initial wash with 10 ml of 1 M KCl, 50 mM Tris base (untitrated, pH 10.3; buffer 3) removed the majority of bound protein, and CNP was then eluted with 5 ml of buffer 3 containing 1 mM adenosine. At this point, aliquots were taken for SDS-PAGE analysis, activity assays, and protein determination. To preserve activity, crystalline bovine serum albumin was added to 5 mg/ml to the remaining sample. The adenosine was removed by 18 h of dialysis with three 1-liter changes of 1 M KCl and 50 mM Tris-HCl, pH 9.0. Without the addition of bovine serum albumin, CNP activity declined during storage at -80 °C. With bovine serum albumin, no loss of activity was detected after 12 months of storage at -80 °C with repeated thawing and freezing.

Molecular Weight Determinations

For SDS-PAGE, the method of Laemmli (15) was followed using 14% separating and 6% stacking gels. Proteins were stained with Coomassie Blue R-250 at 60 °C. For gel-filtration chromatography, a 1.5 50-cm column containing Sephacryl HR200 was used with 1 M KCl buffered with 100 mM Tris-HCl, pH 9.0, at 4 °C at a flow rate of 19 ml/h. Fractions were collected each minute and assayed for CNP activity by assay 1.

Inactivation of CNP by EDTA

A 300-µl aliquot of purified CNP solution containing 5 mg/ml bovine serum albumin was dialyzed for 140 h against three 100-ml volumes of 1 M KCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 9.0, at 4 °C changed at 0, 48, and 96 h. After dialysis, 1% of the starting enzyme activity remained. The EDTA was then removed by size exclusion chromatography using a Bio-Spin 6 Column (Bio-Rad) prewashed with 1 mM EDTA and re-equilibrated with deionized water according the manufacturer's instructions. Nine divalent metal ions (Specpure Analytical Standard salts (Johnson Matthey, Ward Hill, MA)) were added to reaction mixtures for the first stage of assay 2 at concentrations of 1, 10, and 100 µM to test for their ability to reactivate the apoenzyme. Those metals found to reactivate the apoenzyme were examined further to determine their optimal concentrations for reactivation. Percent reactivation of the apoenzyme was calculated as: [(activity of apoenzyme with metal ion addition) minus (activity without metal ion addition)]/[activity of control without EDTA] 100.

Metal Content Determination

ICPES analysis of the purified CNP was performed by the Chemical Analysis Laboratory, University of Georgia (Athens, GA) using the Thermo Jarrell Ash 965 Inductively Coupled Argon Plasma with simultaneous 31 element capability. The CNP was tested for the presence of 31 elements including calcium, cadmium, cobalt, copper, iron, magnesium, manganese, nickel, and zinc which are most frequently found as components of metalloproteins. The metal content of CNP samples was corrected for metal content in control samples containing buffer alone.


RESULTS

Periplasmic Location

Previously, we demonstrated the CNP activity of V. fischeri to be soluble in the periplasm(6) . That study, however, did not address the possibility that the enzyme also occurs in a cytoplasmic membrane- or outer membrane-associated form or that it is released into the extracellular environment. To test these possibilities, we examined membrane and soluble fractions of V. fischeri cells and the cell-free growth medium for CNP activity. Cells were separated into outer-membrane, cytoplasmic membrane, and cytoplasm/periplasm (soluble) fractions. Total cellular CNP activity before fractionation was 291 units, and the fractions were found to contain 0.5, 6, and 224 units, respectively, demonstrating that the enzyme is not associated with either the outer membrane or the cytoplasmic membrane. No CNP activity was observed in the cell-free growth medium following growth of V. fischeri cells to high population density or in protein precipitated from the cell-free growth medium by the addition of ammonium sulfate to 80% of saturation. The enzyme, therefore, does not appear to be released into the extracellular environment.

Purification

A four-step procedure was developed that resulted in purification of the enzyme by approximately 4000-fold (Table 1) and to apparent homogeneity (Fig. 1). The first step, chloroform shock, served to disrupt the outer membrane and release the periplasmic contents from the cells, giving an approximately 3-fold increase in specific activity relative to whole cells. No cell lysis was detected by phase contrast microscopy when cells were mixed gently with the chloroform. Vigorous mixing of cells with the chloroform eliminated most of the CNP activity, as did freezing of the sample at any step in the purification procedure before it was complete.




Figure 1: SDS-PAGE analysis during purification of the V. fischeri periplasmic CNP. PE, periplasmic extract; AS, 40-70% ammonium sulfate fraction; DS, DEAE-Sephacel eluent; BA, Cibacron Blue-agarose eluent; and, PS, protein standards (66 kDa, bovine serum albumin; 45 kDa, ovalbumin; 36 kDa, glyceraldehyde-3-phosphate dehydrogenase; 29 kDa, carbonic anhydrase; 24 kDa, trypsinogen; 20.1 kDa, trypsin inhibitor; 14.2 kDa, -lactalbumin).



The second step, fractionation of the periplasmic suspension with ammonium sulfate, gave a 2-fold increase in specific activity. It also served to concentrate the sample for the two subsequent chromatographic steps, in which most of the purification was achieved.

In the third step, the resolubilized ammonium sulfate fraction was bound to and eluted from DEAE-Sephacel, resulting in a 25-fold purification. Earlier purification attempts using buffer 2 in this anion-exchange step at a pH of 6.0, which is close to the protein's calculated pI of 5.5(7) , were successful but substantial losses of activity occurred. Therefore, the enzyme is apparently unstable at low pH. Adjusting buffer 2 to pH 7.5 (Table 1) eliminated the loss of activity.

In the final step, in which the DEAE-Sephacel eluate was bound to and eluted from Cibacron Blue-agarose, a 26-fold purification was achieved. The simplicity of the purification procedure overall results from the high affinity of the protein for the blue-agarose dye matrix in this step. The high affinity permits the extreme conditions of high KCl concentration and high pH to be used to elute the majority of contaminating proteins. Adenosine, a competitive inhibitor of the V. fischeri CNP with a K of approximately 800 µM (data not shown), was used at a concentration of 1 mM in the wash buffer to elute CNP from the blue-agarose. SDS-PAGE analysis of the adenosine eluate revealed a single protein, even when the gel was heavily loaded (Fig. 1). The enzyme was estimated to account for approximately 0.025% of the total cellular protein and 0.07% of the periplasmic protein. Previously, the enzyme purified in this manner was subjected to microsequence analysis of the first 20 residues at the amino-terminal end, and the sequence for the mature protein matched that deduced from the cloned gene(7) .

Molecular Mass of the Native CNP

The purified protein exhibited a monomeric molecular mass of approximately 34,000 Da on SDS-PAGE (Fig. 1), which is consistent with the mass of the protein calculated from the deduced amino acid sequence of the cloned cpdP gene (33,636 for the protein without its leader sequence; 7). To determine if the enzyme was active as a monomer, we used a calibrated Sephacryl gel filtration column to conduct size exclusion chromatography of the purified protein under non-denaturing conditions. The CNP activity in each of three trials eluted as a single molecular species, with an estimated molecular mass of 32,000 ± 800 Da (mean ± S.D.). A representative experiment is shown in Fig. 2. The similarity of the estimated masses under denaturing and non-denaturing conditions indicates that the native enzyme is active as a monomer and may be present in the periplasm as such.


Figure 2: Native molecular weight determination of V. fischeri periplasmic CNP. One of three experiments is shown in which proteins were separated on Sephacryl HR200. CNP (32.4 kDa, Log molecular weight = 4.51) purified as described in the text () and protein size standards () (160 kDa, aldolase; 106 kDa, 6-phosphogluconate dehydrogenase; 66 kDa, bovine serum albumin; 45 kDa, ovalbumin; 29 kDa, carbonic anydrase; 21 kDa, adenylate kinase).



cAMP Hydrolysis: Kinetics, pH, and Temperature Effects

Activity of the purified periplasmic protein was maximal at pH 8.25, with half-maximal activities at pH values of approximately 7.0 and 9.0 (Fig. 3). The enzyme obeys typical Michaelis-Menten kinetics and has a Kfor cAMP of 73 µM at pH 8.25 and 25 °C (Fig. 4A), as derived from a Hofstee plot(16) . V at 25 °C, calculated from the Hofstee plot (Fig. 4A), was 3700 units/mg protein, and the measured rate at 2 mM cAMP was approximately 3400 units/mg. The corresponding turnover number using the calculated molecular mass of 33,636 is 1.24 10/min.


Figure 3: Effect of pH on hydrolysis of cAMP by V. fischeri periplasmic CNP. Assay 2 was used, and the buffers were 50 mM glycine with 50 mM glycyl-glycine () and 100 mM MES (). Data presented are the averages of duplicate assays. For each data point, the measured values differed by <5% from the average.




Figure 4: Effect of temperature on hydrolysis of cAMP by V. fischeri periplasmic CNP. The assay mixture was adjusted to pH 8.25 at each temperature. Data presented are the averages of duplicate assays. A, Hofstee (16) plots were used to calculate values of K and V for cAMP hydrolysis at various temperatures: 5 °C, K = 53 µM and V = 588 units/mg (); 15 °C, K = 59 µM and V = 2077 units/mg (); 25 °C, K = 73 µM and V = 3730 units/mg (); 35 °C, K = 132 µM and V = 8764 units/mg (). For each data point, the measured values differed by <3% from the average. B, plot of measured specific activities at different temperatures. For each data point, the measured values differed by <5% from the average.



It was of interest to determine if the CNP exhibited maximum activity at a temperature that the bacterium experiences in seawater. Enzymatic reactions in V. fischeri, as in many marine plants and poikilothermic animals, generally occur at temperatures of approximately 27 °C or lower. Remarkably, the rate of the CNP-catalyzed reaction increases with temperature up to 60 °C (Fig. 4B) at which temperature the measured specific activity was about 27,000 units/mg, indicating that the CNP is active at temperatures much higher than it is expected to experience in nature. K and V were determined at 10 °C intervals between 5 and 35 °C (Fig. 4A). The K for cAMP increased from 53 µM at 5 °C to 132 µM at 35 °C, and V increased by 15-fold over the same range, giving a coefficient of 2.47/10 °C rise in temperature.

Substrate Specificity

Previously, we demonstrated that the V. fischeri periplasmic CNP is specific for 3`:5`-cyclic nucleotides(6) . Consistent with that study, cGMP was utilized at approximately half the rate of cAMP (Table 2). The rates observed with cCMP, cIMP, and cUMP were similar to or somewhat higher than that with cAMP, whereas those with the 2`-deoxy-cyclic nucleotides, 2`-deoxy-cAMP and cTMP, were 3-fold lower.



Metal Content

Our earlier observation that EDTA inhibits activity (6) suggested that the CNP might be a metalloenzyme or might require a divalent metal ion cofactor for full activity. Furthermore, sequence similarity of the V. fischeri enzyme to a known zinc enzyme, the low affinity CNP (PDE1) of S. cerevisiae, including the conservation of 4 histidine residues in the two sequences (7) , suggested that the V. fischeri enzyme contains zinc. Therefore, we tested three chelators of zinc for their effects on CNP activity: EDTA, dithiothreitol, and 1,10-orthophenanthroline. The chelators, present in the reaction mixture at 1 mM, inhibited CNP activity 23, 64, and 98%, respectively. With regard to inhibition by dithiothreitol, a strong chelator of zinc(17) , it should be noted that the mature CNP protein has no cysteine residues(7) ; thus, the inhibition by dithiothreitol cannot be due to effects on sulfhydryl groups or disulfide bonds. Subsequent analysis by ICPES detected 2.2 mol zinc/mol CNP, while none of the other 30 elements tested was detected in significant quantities. We conclude that the native V. fischeri CNP is a zinc-containing enzyme, with two atoms of zinc/peptide.

Reactivation of zinc-free apoenzyme with various metal ions supported this conclusion. The apoenzyme was prepared by dialysis of the purified protein against EDTA, followed by size exclusion chromatography to separate EDTAmetal complexes from the apoenzyme. 140 h of dialysis was required to reduce CNP activity to 1% that of a control sample dialyzed against buffer alone, which retained 94% of its original activity (Fig. 5A). After removal of EDTA from the sample, nine divalent metal ions, Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, and Zn, were added to the apoenzyme to test their ability to restore activity. Four metal ions reactivated the apoenzyme to varying degrees (Fig. 5B). Zinc was the most effective; 1 µM restored 94% of the activity of the control sample. Concentrations above 1 µM were less effective or inhibitory. Copper (1 µM), magnesium (10 mM), and calcium (50 µM) restored 33, 47, and 67% of the activity, respectively. Magnesium was still increasing enzyme activity at 10 mM, the highest concentration tested.


Figure 5: Inactivation of the V. fischeri periplasmic CNP by EDTA and reactivation by metal ions. A, activity of CNP dialyzed against buffer containing 1 mM EDTA () and against buffer without EDTA (). B, activity of EDTA-inactivated CNP upon addition of various metal ions (Zn (), Ca (), Cu (), Mg ()). Data presented are the average of duplicate assays. For each data point, the measured values differed by <8% from the average.



To test for the involvement of a loosely bound metal ion in CNP activity, metal ions were added to the reaction mixture with holoenzyme that had not been exposed to EDTA. Of the nine metal ions tested with the apoenzyme, none enhanced activity of the holoenzyme at a concentration of 100 µM, whereas Zn and Cu inhibited holoenzyme activity about 25% and Cd inhibited 77%. No inhibition was observed with calcium or magnesium.


DISCUSSION

Purification of the the periplasmic CNP of V. fischeri to apparent homogeneity permitted the characterization of some of its properties. Several of these, including its narrow substrate specificity, stability, abundance in the cell, specific activity, and the ability of certain metal ions to reactivate apoenzyme are unusual and provide insight into the role of the enzyme in the biology of V. fischeri and the ecology of cyclic nucleotides in seawater.

The narrow substrate specificity of the V. fischeri CNP is atypical for a periplasmic nucleotidase. Other nucleotide degrading periplasmic phosphatases, such as alkaline phosphatase, acid phosphatases, 2`:3`-cyclic phosphodiesterase:3`-nucleotidase, and UDP-glucose hydrolase:5`-nucleotidase, are each capable of hydrolyzing multiple types of phosphorylated compounds, but the CNP of V. fischeri is specific for 3`:5`-cyclic nucleotides; it shows no activity with 2`:3`-cAMP, ADP, ATP, or non-nucleotide phosphate esters (6) . Activity measurements with various 3`:5`-cyclic nucleotides suggest that the 2`-oxygen of the ribose moiety is necessary for full activity of the enzyme, and that the enzyme does not discriminate between purines and pyrimidines (Table 2).

Despite its narrow substrate specificity, the V. fischeri enzyme is similar to many periplasmic phosphatases in containing zinc. Alkaline phosphatase(18) , 2`:3`-cyclic phosphodiesterase:3`-nucleotidase(19) , and UDP-glucose hydrolase:5`-nucleotidase (19) are zinc metalloenzymes. However, there appears to be no significant amino acid sequence similarity between the four proteins. Other than the low affinity CNP (PDE1) of S. cerevisiae, which also contains 2 atoms of zinc/peptide(20) , no eukaryotic CNPs have been shown to be metalloenzymes.

The mature CNP protein has no cysteine residues(7) , and, therefore, no disulfide bonds to stabilize its tertiary structure. Nonetheless, several observations in this report indicate that it is a very stable protein. Full catalytic activity was retained after exposure of the enzyme to 60 °C (Fig. 4B) or to a pH of 10.3 (as used in the dye-affinity step of the purification procedure). In addition, the binding of one or both of the zinc ions is tight, since prolonged dialysis against EDTA was required to inactivate the CNP; after 24 h of dialysis, the activity was 75% of the starting value, and a period of 140 h was required to reduce the activity by 99% (Fig. 5A). A prolonged dialysis against EDTA is also required to inactivate the zinc-containing CNP of S. cerevisiae(20) , and it is possible that the zinc ions in both the yeast CNP and that from V. fischeri are firmly bound to the same ligands (see below).

Copper, magnesium, and, calcium, are all unusual substitutes for zinc, but in metal substitution experiments with the V. fischeri enzyme, activity was partially restored to the apoenzyme by each of these metal ions. Copper has been used as a spectroscopic probe in metal substitution experiments, but with the notable exception of superoxide dismutase, copper-substituted zinc enzymes usually are inactive(21) . Similarly, glyoxylase is one of a few zinc enzymes that is active when substituted with magnesium(22) . Yeast enolase has been shown by x-ray diffraction to bind zinc or calcium at the same site as the native magnesium ion, but the calcium-substituted enzyme is inactive due to an alteration in the conformation of bound substrate (23) . The difficulty in removing zinc from the V. fischeri CNP and the reactivation of the apoenzyme by calcium, which is surprising because calcium has a valence electronic configuration distinct from that of zinc, may indicate that zinc serves as a structural, rather than a catalytic, component in CNP.

However, contrary to the proposal that the zinc of CNP is structural, a recent analysis of zinc enzymes with known structures (24) suggests that the zinc might be catalytic. In that study, it was pointed out that non-catalytic zinc has 2 or more cysteine residues as ligands, of which the mature V. fischeri CNP has none. In addition, catalytic zinc often has 1 or more histidine residues as ligands, of which the CNP has 4 that, by sequence comparison, are evolutionarily conserved(7) . Determination of the role of zinc in the CNP of V. fischeri will require further examination.

The rapid kinetics of the V. fischeri enzyme may make it an interesting enzyme for studies of catalytic mechanism. Using the turnover number and K determined at 25 °C (Fig. 4A), k/K can be calculated to be 2.8 10 sM, which indicates that the rate of formation of the enzyme-substrate complex is nearly as fast as allowed by diffusion controlled encounters between CNP and cAMP(25) . At 30 °C, the assay temperature used in studies of other CNPs, the V. fischeri enzyme has a specific activity of 7100 units (Fig. 4B), a value more than 20 times greater than any previously reported for a CNP that is specific for 3`:5`-cyclic nucleotides, including that of the extracellular CNP of D. discoideum(26) and the low affinity CNP of S. cerevisiae(20) to which it has substantial amino acid sequence identity(7) . The highest previously reported specific activity for a CNP is that from bovine brain and heart, 300 units/mg(27) . The highest specific activity for catalysis of cAMP hydrolysis by a CNP from a bacterium other than V. fischeri is that from Serratia marcescens, 295 units/mg(28) .

The pH/activity profile (Fig. 3) supports the involvement of 1 or more histidine residues in CNP activity. Although side chain ionizable groups on amino acid residues in proteins often have pK values that are different from those seen with free amino acids, and the ionizable groups involved in enzyme catalysis cannot be definitively identified from a pH/activity profile, such profiles can be suggestive, especially if additional information about the enzyme's structure and function is available. The V. fischeri CNP exhibits half-maximal activity at pH values of 7 and 9. Imidazole side chains of histidine residues have pK values in the range of 5-8(29) , and 4 histidines, which occur infrequently in bacterial proteins, are conserved in the CNPs of V. fischeri, S. cerevisiae, and D. discoideum(7) . Such highly conserved residues often have essential roles in enzyme structure and/or catalysis. Thus, we assume that further study may show that 1 or more of the histidines in V. fischeri CNP must be in the unprotonated state for most efficient catalysis. Likewise with regard to the half-maximal activity at pH 7, zinc-bound water is a participant in many hydrolytic reactions, and it ionizes with a pK of about 7. Our data are also consistent with a role for zinc-bound water in the activity of CNP. The implications of the half-maximal activity at pH 9 are less clear. Protein -amino groups, lysine -amino groups, and tyrosine phenolic hydroxyl groups generally ionize with pK values of about 9, and our results (Fig. 3) might indicate involvement of one or more of these groups in the CNP-catalyzed hydrolysis of cAMP.

The biochemical characteristics of the V. fischeri CNP are consistent with hypotheses for the biological role of the enzyme in the ecology and symbiosis of V. fischeri. The enzyme has been proposed to degrade cAMP free in the environment(6) . Many organisms release cAMP(30, 31) , but the concentration in the environment is low (31, 32) . The CNP of V. fischeri, because of its periplasmic location and high specific activity, could function in a manner analogous to phosphate scavenging periplasmic enzymes in bacteria. Many bacteria have periplasmic 5`-nucleotidase activity, which degrades AMP to inorganic phosphate and adenosine prior to their transport into the cytoplasm and subsequent metabolism. The V. fischeri CNP, therefore, represents a novel class of enzyme that can catalyze the first step in the recovery of cAMP from the environment.

In the luminescent mutualism of V. fischeri with sepiolid squid and monocentrid fish(33, 34) , the enzyme has been hypothesized to permit V. fischeri, exclusively, to utilize host-provided cAMP as a source of nutrition. According to the hypothesis, V. fischeri cells in the animal light organ elicit the overproduction and release of host cAMP by secreting a toxin that, through an ADP-ribosylating activity, interferes with the regulation of host adenylate cyclase in a manner analogous to the secretion and activity of cholera enterotoxin by V. cholerae in the human intestine (6, 7, 35) . Recent evidence indicating that V. fischeri contains toxRS genes (36) is consistent with this hypothesis. Nonetheless, a direct test of the symbiosis hypothesis will require examination of the ability of a cpdP null mutant of V. fischeri to colonize its animal host.


FOOTNOTES

*
This work was supported by National Science Foundation Grants MCB 91-04653 and 94-08266 (to P. V. D.) and by a grant from the Endeavour Foundation (to N. W. C.). This is contribution no. 8953 from the Woods Hole Oceanographic Institution. 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: Biology Dept., Redfield Laboratory, 45 Water St., Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1049. Tel.: 508-289-3209; Fax: 508-457-2195; E mail: pdunlap@whoi.edu.

The abbreviations used are: CNP, 3`:5`-cyclic nucleotide phosphodiesterase; ICPES, inductively coupled plasma emission spectroscopy; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank R. Auxier of the Chemical Analysis Laboratory, University of Georgia, for conducting the ICPES metal content analysis, L. Ball of the Inductively Coupled Plasma Facility at the Woods Hole Oceanographic Institution for technical assistance and for providing the Specpure Analytical Standard salts, and L. Gilson for comments on the manuscript.


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