Isolation and Mapping of Polynucleotide Phosphorylase Mutants of Escherichia coli

Three strains of Escherichia coli with altered polynucleotide phosphorylase, Q7, Q13, and Q27, were isolated by screening clones from heavily mutagenized cultures for low levels of the enzyme. The three mutations were found to cotransduce with argG and asp, and the pnp locus which they define was mapped with respect to these loci. An explanation for the nonreciprocal cotransduction frequencies observed with asp is provided by the demonstration of an unlinked asp-suppressing locus.     

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.     

Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens]

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.     

Thermostable polynucleotide phosphorylases from Bacillus stearothermophilus and Thermus aquaticus.

Polynucleotide phosphorylase from Bacillus stearothermophilus has been purified to homogeneity. Polyacrylamide gel electrophoresis run under denaturing conditions indicates that the enzyme is a tetramer with subunits of apparent molecular weight 51,000 daltons. A partial purification of polynucleotide phosphorylase from Thermus aquaticus has also been effected. The two enzymes show similar catalytic properties, which differ little from those of mesophilic polynucleotide phosphorylases. The use of thermostable polynucleotide phosphorylases for in vitro nucleic acid synthesis is discussed.     

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.     

A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus

We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second activity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed.     

The effect of trypsin digestion on the activities of polynucleotide phosphorylase

1. Treatment of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) with trypsin causes a preferential loss of its cytidine diphosphate and uridine diphosphate polymerization activities. 2. The phosphorolytic activity of the enzyme towards polycytidylic acid is unaffected in conditions in which the cytidine diphosphate-polymerization activity without added primer is virtually abolished. 3. The treated enzyme retains its altered pattern of activities when purified fivefold by gel filtration. 4. The effect on the cytidine diphosphate-polymerization activity is due, in part, to a large increase in primer requirement as a result of proteolysis, and is qualitatively independent of the state of purity of the polynucleotide phosphorylase. 5. The enzyme is protected from trypsin degradation by nucleic acids, polynucleotides and nucleoside disphosphates. 6. A similar, but less marked differential effect, is caused by alpha-chymotrypsin.     

Polynucleotide Phosphorylase Has a Role in Growth of Escherichia coli

Mutants having low levels of polynucleotide phosphorylase activity grow poorly at 45 C. All revertants isolated for their ability to grow better at that temperature also regained higher levels of polynucleotide phosphorylase and the ability to be induced for tryptophanase. Thus, a physiological role is implied for the enzyme polynculeotide phosphorylase.     

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization.A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity.Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.     

Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.     

Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPase-deficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite- and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.     

Production of RNA by a polymerase protein encapsulated within phospholipid vesicles

Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.Abbreviations ADP adenosine diphosphate - DMPC dimyristoyl phosphatidylcholine - EDTA ethylenediaminetetraacetic acid - LUV large unilamellar vesicle - MLV multilamellar vesicle - PAGE polyacrylamide gel electrophoresis - PNPase or PNP polynucleotide phosphorylase - SUV small unilamellar vesicle Correspondence to.: A.C. Chakrabarti     

Wine yeast strains engineered for glycogen overproduction display enhanced viability under glucose deprivation conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase.

It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.     

Synthesis of Polynucleotides by Microorganisms

Polynucleotides could be synthesized from nucleoside diphosphates by microorganisms belonging to genera Pseudotnonas, Serratia, Xatuhonwnas, Proteus, Aerobacter, Bacillus, and Brevibacterium. These strains were rich in polynucleotide phosphorylase easily extractable from cells and poor in both nuclease and nucleoside-diphosphate-degrading enzymes. Polynucleotide phosphorylase was effectively extracted from the bacterial cells, that had been once soaked in saturated saline solution, with hypotonic solution. Synthesis of polynucleotides was observed not only when the substrates were incubated with polynucleotide phosphorylase preparation isolated from the bacterial cells, but also when the substrates were added directly to the bacterial cultures.     

Mutants of Escherichia coli with altered hydrogenase activity

Mutant strains of Escherichia coli which expressed different levels of hydrogenase activity when grown anaerobically under a variety of conditions were obtained by mutagenesis and selective growth and screening procedures. Four classes of mutants were isolated, ranging from those devoid of enzyme activity to those expressing maximal activity under all growth conditions. One class of mutants (A) could not grow on fumarate plus H2 in the presence of active fumarate reductase. Since hydrogenase is essential for growth under these conditions some of these strains may be hydrogenase-negative. Three other classes of mutants were isolated which were all hydrogenase-positive and fully expressed this activity when grown on fumarate plus H2. They differed in the level of expression of hydrogenase activity when grown anaerobically on glucose, conditions which do not require hydrogenase for growth. Class B mutants expressed less activity, while class C mutants expressed more activity than the parental strain. Class D mutants fully expressed hydrogenase activity and were dependent on the enzyme for growth. The different strains were also assayed for reduction of dyes by hydrogen and for evolution of hydrogen from reduced methyl viologen. Some of the hydrogenase-positive strains showed altered activities in these assays suggesting that mutations may have occurred either in enzymes or proteins required for reaction with dyes or in the hydrogenase enzyme itself.     

The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate-polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.     

Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Properties of the enzyme*

A modified purification is described for an enzyme, from Escherichia coli B, which polymerizes deoxyribonucleoside-5′ diphosphates. Under appropriate conditions, the enzyme will add a single deoxyribonucleotide residue to a deoxyribo-oligonucleotide primer. At all stages, the enzyme activity copurified with the activity which will polymerize adenosine-5′ diphosphate (polynucleotide phosphorylase). Studies of heat stability, the effect of various temperatures of reaction and of disc gel electrophoresis failed to provide evidence that the two activities are separable.     

Enzymatic synthesis of deoxyribo-oligonucleotides of defined sequence. Deoxyribo-oligonucleotide synthesis*

An enzyme, which is probably identical with polynucleotide phosphorylase, was prepared from Escherichiacoli B. In the presence of Mn(2+) it catalyzes the addition of one (and to a slight extent more) residue of deoxyribonucleotide residue from the diphosphate to an oligodeoxyribonucleotide primer. The shortest effective primers contained three phosphate residues. Ribodinucleotides were effective as primers and accepted two or three deoxyribonucleotide residues under these conditions. The application of the procedures to the convenient synthesis of certain defined oligodeoxyribonucleotides up to nine residues long is discussed.