Ecto-5’-nucleotidase: Structure function relationships

Ecto-5'-nucleotidase (ecto-5'-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5'-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5'-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5'-NT is reviewed in relation to the catalytic properties and enzyme function.     

ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5'-nucleotidase. Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity.

IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5'-nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molecular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 M promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C-terminus caused profound kinetic and structural changes, including a 29-fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5'-nucleotidase subunits.     

Complete deficiency of 5'-nucleotidase activity in Escherichia coli leads to loss of growth on purine nucleotides but not of their excretion

Escherichia coli has many periplasmic phosphatase activities. To test whether it can take up and excrete purine nucleotides, we attempted to completely disrupt periplasmic 5'-nucleotidase activity. A 5'-nucleotidase activity was induced in ushA knockout mutant cells, which lack major 5'-nucleotidase activity, when they were grown with purine nucleotides as the sole carbon source. Using DNA macroarrays to compare global gene expression in wild-type and ushA knockout mutant cells cultured with IMP or GMP as the sole carbon source, we identified two genes that were induced in the ushA knockout mutant cells and encoded signal sequence needed for secretion. One of the genes, aphA, encoded a 5'-nucleotidase activity and was induced by IMP or inosine. An ushA aphA double knockout mutant was shown to be unable to grow on purine nucleotides as the sole carbon source. To investigate the excretion of purine nucleotides, we constructed an ushAaphA double knockout mutant of an inosine-producing strain and found that it accumulated IMP in the medium. In addition, when the guaBA operon was introduced into the ushAaphA double knockout IMP producer, GMP was released into the medium. These observations imply the existence of efflux activity for purine nucleotides in E. coli.     

Sequence analysis of nutA gene encoding membrane-bound Cl(-)-dependent 5'-nucleotidase of Vibrio parahaemolyticus

The membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus is unique in requiring Cl- for activity. We cloned the nutA gene encoding the 5'-nucleotidase and sequenced it. It contained an open reading frame consisting of 1,680 nucleotides capable of encoding a protein of 560 amino acid residues. The first 21 amino acid residues of the N-terminal portion of this protein seem to be a signal peptide. The rest of the polypeptide (539 residues) is hydrophilic, and its molecular weight was calculated to be 60,008, which is in good agreement with the value of 63 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the 5'-nucleotidase derived from the cloned nutA gene. We tried to determine the amino acid sequence of the N-terminal portion of the purified enzyme. However, the N-terminal residue seemed to be blocked. As this 5'-nucleotidase can be solubilized from membrane vesicles with detergent, it may be a lipoprotein. The amino acid sequence around the possible cleavage site of the 5'-nucleotidase had homology with the sequences of the cleavage sites of the lipoproteins of Escherichia coli and other bacteria. The amino acid sequence had high (about 60%) homology with the sequence of periplasmic 5'-nucleotidase (uridine diphosphate sugar hydrolase, the product of the ushA gene) of E. coli. It also contained regions that showed some homology with the nucleotide binding sites of many nucleotide binding proteins.     

Ultrastructural localization of 5'-nucleotidase in guinea pig neutrophils based upon the use of cerium as capturing agent

A cytochemical method for 5'-nucleotidase localization in which cerium serves as the capture agent in order to enzymatically detect liberated inorganic phosphate has been developed. The method has been established in cell-free model systems and in guinea pig neutrophils where 5'-nucleotidase is restricted to the plasmalemma as an ectoenzyme. This cerium-based method gives better results for ultrastructural localization of 5'-nucleotidase than conventional lead-based methods.     

Membrane-bound 5'-nucleotidase in marine luminous bacteria: biochemical and immunological properties

A novel 5'-nucleotidase previously described in halophilic Vibrio costicola was detected in marine Vibrio and Photobacterium strains. The enzyme of marine bacteria was similar in its properties to the 5'-nucleotidase of Vibrio costicola; it was outwardly oriented in the cytoplasmic membrane and dephosphorylated nucleoside 5'-tri-, di-, and mono-phosphates to respective nucleosides before uptake. The enzyme in marine strains was immunologically cross-reactive with the antibody raised against the purified 5'-nucleotidase of Vibrio costicola. The uptake of the products of ATP hydrolysis was studied in Vibrio harveyi, and it was shown that both adenosine and inorganic phosphate released upon the action of 5'-nucleotidase were rapidly taken up by the cell.     

Purine catabolism in man: inhibition of 5'-phosphomonesterase activities from placental microsomes.

The 5'-phosphomonoesterase activity of 5'-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.5) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5'-nucleotidase suggest a sequential mechanism of interaction between AMP nad MgCl2, with a Km of 14 and 3 muM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhititors when AMP was the substrate, with a Ki slope of 120 muM. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. These observations suggest that 5'-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5'-nucleotidase.     

Mutants of Escherichia coli K-12 "cryptic," or deficient in 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (cyclic phosphodiesterase) activity

Mutants of Escherichia coli have been selected for the absence of 5'-nucleotidase (uridine diphosphate-sugar hydrolase) and 3'-nucleotidase (2',3'-cyclic phophodiesterase). Mutants selected for the absence of 5'-nucleotidase are of two kinds: those that lack detectable activity for the enzyme (Ush(-)), and those that possess activity when cell extracts are assayed, but not when intact cells are assayed (cryptic; Crp(-)). The latter class is probably identical to a type of mutant previously reported by Ward and Glaser. When mutants are selected for the absence of 3'-nucleotidase, Crp(-)mutants are also obtained. Thus far, however, mutants totally lacking this enzyme have not been found. The location on the genetic map of one ush mutation is at position 11 min and that of one crp mutation at approximately 67 min. In the crp mutant, 5'-nucleotidase and 3'-nucleotidase remain located in the periplasm. This mutant is also cryptic for alkaline phosphatase but not for acid hexose phosphatase. Treatment of cells with ethylenediamine-tetraacetate substantially alleviated crypticity. These data are discussed in terms of the organization of periplasmic enzymes and of the outer membrane as a permeability barrier.     

A microtiter plate assay for inorganic phosphate

A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca2+ + Mg2+)ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate.     

CYTOCHEMICAL LOCALIZATION OF 5''-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER : I. The Origin of 5''-Nucleotidase Activity in Microsomes

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO₃)₂ and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb²⁺, or Pb²⁺ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb²⁺, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb²⁺ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb²⁺ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.     

Some properties of human erythrocyte pyrimidine 5'-nucleotidase

In haemolysates human erythrocyte pyrimidine 5'-nucleotidase had a single optimum at pH 7.2 with CMP and 6.75 with UMP as substrate. The purified enzyme showed two pH optima at pH 6.25 and 7.2 with UMP as substrate. The enzyme was inhibited by both its products - inorganic phosphate and pyrimidine nucleoside. The inhibition by inorganic phosphate appeared to be non-competitive with Ki = 1.5 mM. Contrary to previous reports adenosine and inosine did not inhibit the enzyme.     

Biochemical and Cytochemical Evidence for the Polar Concentration of Periplasmic Enzymes in a “Minicell” Strain of Escherichia coli

A number of "surface" enzymes of Escherichia coli (i.e., among those selectively released by osmotic shock) all displayed higher specific activities in extracts of minicells than in extracts of typical rod forms; these enzymes included alkaline phosphatase, cyclic phosphodiesterase, acid hexose monophosphatase, 5'-nucleotidase, and ribonuclease I. In addition, alkaline phosphatase, cyclic phosphodiesterase, and acid hexose monophosphatase were cytochemically localized to regions of minicell periplasm that resembled reactive polar enlargements of the periplasm in rod forms. In contrast, a number of "internal" cytoplasmic enzymes (inorganic pyrophosphatase, beta-galactosidase, glutamine synthetase, polynucleotide phosphorylase, and ribonuclease II) showed elevated or similar specific activities in extracts of rod forms versus extracts of minicells. A specific heat-labile inhibitor for 5'-nucleotidase, known to occur in the cytoplasm, also showed no enrichment in minicells. These findings indicate that the "surface" enzymes are segregated in vivo into the terminal minicell buds, possibly because these enzymes are concentrated in the polar enlargements of the periplasm in typical rod forms.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis

1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.     

Hydrolysis of 5'-phosphonates and nucleoside phosphates by phosphatases of various origin and human and calf serum]

The hydrolysis of 5'-phosphonates of 2'-deoxythymidine and its 3'-modified analogs, inhibiting the HIV reproduction, by the E. coli alkaline, calf intestine and human placenta phosphatases as well as by the Crotalus atrox venom 5'-nucleotidase were studied. Transformations of 5'-phosphonates of adenosine and its analogs during incubation with human and fetal calf blood sera were investigated. The nucleotide derivatives modified at the phosphate residue were not hydrolyzed by any of the phosphatases studied except for the cobra venom 5'-nucleotidase, the effectiveness of the latter depended on the substitutes at both phosphate and sugar residues. 2'-Deoxyadenosine incubation with blood sera resulted in its transformation to 2'-deoxyinosine and then to hypoxanthine. 2'-Deoxyadenosine 5'-phosphonates were stable during incubation with blood sera under the same conditions.     

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.     

Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.     

The intracellular localization of Pseudomonas aeruginosa lectins

The localization of the Pseudomonas aeruginosa lectins (PA-I and PA-II) was studied using methods of osmotic shock, freezing and thawing and spheroplast formation. Very slight release of the two lectins occurred when P. aeruginosa was exposed to magnesium-osmotic shock or was frozen and thawed. Under these conditions, release of the periplasmic 5'-nucleotidase occurred, whereas no release of cytoplasmic glucose-6-phosphate dehydrogenase activity was detected. Formation of spheroplasts from P. aeruginosa by gradual removal of the bacterial envelopes revealed low lectin activity in the treatment fluids. Osmotic shock treatment of the lysozyme treated mureinoplasts resulted in low release of glucose-6-phosphate dehydrogenase and the two lectins (10-13%) and a considerable activity (38.4%) of 5'-nucleotidase. The presence of the lectins on the outer and the cytoplasmic membranes enabled intact cells and spheroplasts of P. aeruginosa to agglutinate papain-treated human erythrocytes. These results indicate that the two lectins are located mainly in the cytoplasm with small fractions on the cytoplasmic and outer membranes and in the periplasmic space.