Deoxynucleoside-sensitive mutants of Salmonella typhimurium

Summary Thymineless mutants ofSalmonella typhimurium which are able to grow with low added concentrations of thymine (20 M) fall into two classes on the basis of growth on deoxyribose as sole carbon source. Those which can grow are deoxyribomutase negative and those which cannot are deoxyriboaldolase negative. The former class are inhibited by deoxynucleosides and this provides a method for discriminating between different classes oftlr mutants ofEscherichia coli K12, which cannot utilize deoxyribose as a carbon source. It is suggested that the sensitivity of deoxyriboaldolase negative strains is due to the accumulation of deoxyribose-5-phosphate. The data also indicate that deoxyribose-5-phosphate is the inducer of thymidine phosphorylase. It seems that one or both of the deoxyribose phosphates is the toxic compound, and that reversal of inhibition by ribonucleosides is due to inhibition of the enzymes catalysing their formation from deoxynucleosides. We propose that the symbolsdrm anddra be used to denote the structural genes for deoxyribomutase and deoxyriboaldolase respectively.     

Induction of pyrimidine nucleoside metabolizing enzymes in E. coli B

Induction studies on pyrimidine metabolizing enzymes in E. coli B have shown that the enzymes fall into three distinct groups according to their induction pattern. a) Cytidine deaminase and uridine phosphorylase, are induced by cytidine, CMP and adenosine; no induction was observed with uridine and AMP; b) thymidine phosphorylase is induced by cytidine, adenosine, all deoxyribonucleosides, CMP, deoxyribonucleotides, deoxyribose and deoxyribose-1-phosphate; c) uridine-cytidine kinase, uracil phosphoribosyltransferase, 5'-nucleotidase, thymidine kinase, are uninducible enzymes. Simultaneous addition of cytidine and glucose partially overcomes the cytidine deaminase and uridine phosphorylase induction. Cytidine deaminase reaches its maximum activity levels, in E. coli growing cells in presence of cytidine, two hours before the uridine phosphorylase activity. Maximum glucose repression of cytidine deaminase and uridine phosphorylase was obtained in correspondence of maximum cytidine induction.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

2-Deoxyribose Gene-Enzyme Complex in Salmonella typhimurium: Regulation of Phosphodeoxyribomutase

Phosphodeoxyribomutase, the enzyme which catalyzes the interconversion of 2-deoxyribose-1-phosphate to 2-deoxyribose-5-phosphate, has been partially purified from Salmonella typhimurium. The enzyme had an absolute requirement for manganese ion and was stimulated by glucose-1, 6-diphosphate. Phosphodeoxyribomutase was induced by deoxyribose-5-phosphate and was coordinately regulated with the enzymes thymidine phosphorylase and deoxyribose-5-phosphate aldolase, type II. Mutants deficient in these three enzymes were isolated and mapped close to the threonine locus in S. typhimurium. The three enzymes thymidine phosphorylase, deoxyribose-5-phosphate aldolase, type II, and phosphodeoxyribomutase are controlled by a series of linked genes and appear to constitute an operon.     

Regulation of Thymidine Metabolism in Escherichia coli K-12: Evidence That at Least Two Operons Control the Degradation of Thymidine

In Escherichia coli K-12, the rise in activity of thymidine phosphorylase, phosphodeoxyribomutase, and deoxyribose-5-phosphate aldolase caused by exogenous thymidine is dependent on the synthesis of new enzyme protein. Phosphodeoxyribomutase is induced by the purine ribonucleosides adenosine and guanosine, whereas the other two enzymes are not. The mutase activity induced by thymidine and by the purine ribonucleosides has been shown to be the same enzyme by four different criteria. This independent induction of phosphodeoxyribomutase suggests that the gene for this enzyme is in an operon different from the one that may contain the genes for thymidine phosphorylase and deoxyribose-5-phosphate aldolase.     

Regulation of the deo operon in Escherichia coli: the double negative control of the deo operon by the cytR and deoR repressors in a DNA directed in vitro system.

Summary The synthesis of the four enzymes of the deo operon in Escherichia coli is known from in vivo experiments to be subject to a double negative control, exerted by the products of the cytR and deoR genes.A DNA-directed in vitro protein synthesizing system makes the deo enzymes (exemplified by thymidine phosphorylase) in agreement with in vivo results. Enzyme synthesis is stimulated by cyclic AMP and repressed by the cytR and deoR gene products. Repression by the cytR repressor is reversed by cytidine or adenosine in the presence of cyclic AMP, while repression by the deoR repressor is reversed by deoxyribose-5-phosphate.Assays for the presence of the cytR and deoR repressors were established by use of S-30 extracts prepared from the regulatory mutants.Dissociation constants for repressor-operator binding as well as for repressor-inducer interactions have been estimated from the results.Abbreviations and Symbols deoA (previously designated tpp) Genes coding for: thymidine, phosphorylase - deoB (previously designated drm) deoxyribomutase - deoC (previously designated dra) deoxyriboaldolase - deoD (previously designated pup) purine nucleoside phosphorylase - udp uridine phosphorylase - cytR regulatory gene for cdd, udp, deoC, deoA, deoB, and deoD - deoR (previously designated nucR) regulatory gene for deoC, deoA, deoB, and deoD Enzymes (EC 2.4.2.1) Purine nucleoside phosphorylase or purine nucleoside: orthophosphate(deoxy)ribosyltansferase - (EC 2.4.2.4) thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - (EC 2.4.2.3) uridine phosphorylase or uridine: orthophosphate ribosyltransferase - (EC 4.1.2.4) deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - (EC 2.7.5.6) phosphodeoxyribomutase The deo operon is defined as the gene cluster consisting of deoC deoA deoB deoD. The deo enzymes are the four enzymes encoded by the four genes of the deo operon. cAMP: cyclic adenosine 3,5-monophosphate. CRP: cyclic AMP receptor protein. dRib-5P: deoxyribose-5-phosphate. THUR: 3,4,5,6-tetrahydrouridine; EDTA: ethylene-diamine-tetra-acetate.     

Regulation of Thymidine Metabolism in Escherichia coli K-12: Studies on the Inducer and the Coordinateness of Induction of the Enzymes

A study was made of the regulation of three enzymes that act sequentially in the metabolism of thymidine in Escherichia coli K-12. Under a variety of conditions, two of the enzymes, thymidine phosphorylase and deoxyribose-5-phosphate aldolase, were found to be synthesized coordinately. However, the third enzyme, phosphodeoxyribomutase, was synthesized noncoordinately with the other two enzymes under the same conditions. In addition, the mutase could be fully induced, whereas basal levels of the phosphorylase and the aldolase were maintained. These findings indicate that two operons comprise the genes concerned with the reversible pathway leading from thymidine to acetaldehyde and glyceraldehyde-3-phosphate. In addition to thymidine, it was found that acetaldehyde was an external inducer of these enzymes. The results of induction experiments performed on wild-type cells and mutants defective in the mutase or the aldolase, with thymidine or acetaldehyde as exogenous inducers, strongly suggest that deoxyribose-5-phosphate is more proximal to the intracellular inducer than is thymidine, deoxyribose-1-phosphate, or acetaldehyde.     

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitt's lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.     

Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases

Substrate-stabilization of uridine phosphorylase (uridine:orthophosphate ribosyltransferase, EC 2.4.2.3), thymidine phosphorylase (thymidine:orthophosphate deoxyribosyltransferase, EC 2.4.2.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) from Escherichia coli was investigated by heat-inactivation experiments. Nucleoside substrates stabilized uridine phosphorylase and purine-nucleoside phosphorylase, but not thymidine phosphorylase. Aglycone substrates stabilized only uridine phosphorylase. Phosphate or pentose-1-phosphate ester substrates stabilized all three enzymes. The appropriate pentose-1-phosphate ester was a more effective stabilizer than was phosphate with all three enzymes. In previous reports dealing with the kinetic analysis of these phosphorylases, sequential mechanisms were proposed. Each enzyme appeared to have different sequence of substrate addition. The substrate-stabilization patterns reported here are consistent with the proposed mechanisms.     

2-Deoxyribose Gene-Enzyme Complex in Salmonella typhimurium I. Isolation and Enzymatic Characterization of 2-Deoxyribose-Negative Mutants

Salmonella typhimurium was found to utilize 2-deoxyribose as a sole carbon and energy source. Cells grown in the presence of deoxyribose contained increased levels of deoxyribose kinase, thymidine phosphorylase, and two forms of deoxyribose-5-phosphate aldolase (DR5P aldolase). One form of DR5P aldolase was induced by deoxyribose and coordinately regulated with deoxyribose kinase. The second form of DR5P aldolase was induced by deoxyribose-5-phosphate and coordinately regulated with thymidine phosphorylase. Mutants unable to ferment deoxyribose have been isolated and shown to be lacking either deoxyribose kinase or deoxyribose permease, but none has been found from which DR5P aldolase is missing. Thymine-requiring mutants which are able to grow on low levels of thymine have been isolated and shown, in some cases, to be lacking one or both DR5P aldolases.     

The dgt gene of Escherichia coli facilitates thymine utilization in thymine-requiring strains

The Escherichia coli dGTP triphosphohydrolase (dGTPase) encoded by the dgt gene catalyses the hydrolysis of dGTP to deoxyguanosine and triphosphate. The recent discovery of a mutator effect associated with deletion of dgt indicated participation of the triphosphohydrolase in preventing mutagenesis. Here, we have investigated the possible involvement of dgt in facilitating thymine utilization through its ability to provide intracellular deoxyguanosine, which is readily converted by the DeoD phosphorylase to deoxyribose-1-phosphate, the critical intermediate that enables uptake and utilization of thymine. Indeed, we observed that the minimal amount of thymine required for growth of thymine-requiring (thyA) strains decreased with increased expression level of the dgt gene. As expected, this dgt-mediated effect was dependent on the DeoD purine nucleoside phosphorylase. We also observed that thyA strains experience growth difficulties upon nutritional shift-up and that the dgt gene facilitates adaptation to the new growth conditions. Blockage of the alternative yjjG (dUMP phosphatase) pathway for deoxyribose-1-phosphate generation greatly exacerbated the severity of thymine starvation in enriched media, and under these conditions the dgt pathway becomes crucial in protecting the cells against thymineless death. Overall, our results suggest that the dgt-dependent pathway for deoxyribose-1-phosphate generation may operate under various cell conditions to provide deoxyribosyl donors.     

Demonstration of pyrimidine nucleoside phosphorylases in breast cancer

Two pyrimidine phosphorylase activities have been isolated from the cytosol of cultivated MCF-7 cells of a human breast cancer, by ion exchange chromatography. Both enzymes are responsible for the cleavage of thymidine into thymine and deoxyribose-1-phosphate, for the synthesis of thymidine and for the transfer of deoxyribose from d-uridine to thymine. These activities are likely to participate in the regulation of the pool of pyrimidine nucleosides required for DNA synthesis.     

Thymidine sensitivity of certain strains of Escherichia coli K12.

Summary In studies on thymineless death in Escherichia coli K12, it was noted that certain thymine requiring mutants were inhibited by thymidine. The pattern of inhibition varied with the conditions and media employed. Accumulation of deoxyribose-5-phosphate as a possible reason for inhibition is ruled out since the strains are deoB ^- (formerly drm ^-) and synthesize deoxyriboaldolase constitutively. We report this inhibition to alert investigators who study thymidine metabolism or use thymidine to label the DNA.     

Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.     

Mutants of Escherichia coli unable to metabolize cytidine: isolation and characterization

Summary Strains of Escherichia coli have been selected, which contain mutations in the udk gene, encoding uridine kinase. The gene has been located on the chromosome as cotransducible with the his gene and shown to be responsible for both uridine and cytidine kinase activities in the cell.An additional mutation in the cdd gene (encoding cytidine deaminase) has been introduced, thus rendering the cells unable to metabolize cytidine. In these mutants exogenously added cytidine acts as inducer of nucleoside catabolizing enzymes indicating that cytidine per se is the actual inducer.When the udk, cdd mutants are grown on minimal medium the enzyme levels are considerably higher than in wild type cells. Evidence is presented indicating that the high levels are due to intracellular accumulation of cytidine, which acts as endogenous inducer.Abbreviations and Symbols FU 5-fluorouracil - FUR 5-fluorouridine - FUdR 5-fluoro-2'deoxyuridine - FCR 5-fluorocytidine - FCdR 5-fluorodeoxycytidine - THUR 3, 4, 5, 6-tetrahydrouridine - UMP uridine monophosphate - CMP cytidine monophosphate - dUMP deoxyuridine monophosphate. Genes coding for: cytidine deaminase - edd uridine phosphorylase - udp thymidine phosphorylase - tpp purmnucleoside phosphorylase - pup uridine kinase (=cytidine kinase) - udk UMP-pyrophosphorylase - upp. CytR regulatory gene for cdd, udp, dra, tpp, drm and pup Enzymes EC 2.4.2.1 Purine nucleoside phosphorylase or purine nucleoside: orthophosphate (deoxy)-ribosyltransferase - EC 2.4.2.4 thymidine phosphorylase or thymidine: orthophosphate deoxyribosyltransferase - EC 2.4.2.3 uridine phosphorylase or uridine: orthophosphate ribosyltransferase - EC 3.5.4.5 cytidine deaminase or (deoxy)cytidine aminohydrolase - EC 4.1.2.4 deoxyriboaldolase or 2-deoxy-D-ribose-5-phosphate: acetaldehydelyase - EC 2.4.2.9 UMP-pyrophosphorylase or UMP: pyrophosphate phosphoribosyltransferase - EC 2.7.1.48 uridine kinase or ATP: uridine 5-phosphotransferase     

Pyrimidine deoxyribonucleotide metabolism in Acholeplasma laidlawii B-PG9.

Extracts of Acholeplasma laidlawii B-PG9 were examined for the enzymes associated with the interconversion of the pyrimidine deoxyribonucleotides and the biosynthesis of thymidine nucleotides. A. laidlawii B-PG9 possessed deaminases for deoxycytidine and dCMP, pyrophosphatases for dUTP, phosphorylases for thymidine and uridine, and a membrane-associated pyrimidine deoxyribonucleoside monophosphate phosphatase activity. The role these enzyme activities have in the generation of deoxyribose-1-phosphate during growth may explain the ability of A. laidlawii B-PG9 to utilize either thymine or thymidine for biosynthesis.     

Upregulation of platelet derived endothelial cell growth factor/thymidine phosphorylase by interferon alpha

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine to thymine and deoxyribose-1-phosphate. TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. TP is known to be regulated by several cytokines and interferons. In our HT29 cell line the TP mRNA and activity expression increased 2-3 fold after treatment with interferon alpha.     

Optimum Induction of Recombinant Thymidine Phosphorylase and Its Application

Recombinant E. coli pDEOA was constructed and lactose can be used instead of IPTG to induce the expression of thymidine phosphorylase by pDEOA. The use of lactose at concentrations higher than 0.5 mmol/L had an induction effect similar to that of IPTG but resulted in a longer initial induction time and better cell growth. The thymidine phosphorylase induced by lactose was very stable at 50°C. Intact pDEOA cells induced by lactose can be used as a source of thymidine phosphorylase. Under standard reaction conditions, several deoxynucleosides were effectively produced from thymidine.     

Synthesis of 2-chloro-2'-deoxyadenosine by microbiological transglycosylation using a recombinant Escherichia coli strain

Cladribine (2-chloro-2'-deoxyadenosine) was synthesized using intact cells of the recombinant Escherichia coli strain producing Geobacillus stearothermophilus B-2194 thermostable purine-nucleoside phosphorylase II (EC 2.4.2.1). Use of the cells containing this thermostable enzyme allowed the process to be conducted at a temperature of 70 degrees C, which provided the maximal concentrations of sparingly soluble substrates. The best results were obtained with 2-chloroadenine as a modified base. The highest yield of the target 2-chloro-2'-deoxyadenosine (up to 95% in the case of deoxyguanosine) was reached when using 2'-deoxypurines as donors of deoxyribose. Use of thymidine for these purposes required its considerable molar excess over 2-chloroadenine (up to 6 : 1), which is connected with a nonoptimal amount of endogenous thymidine phosphorylase, necessary for synthesis of deoxyribose-1-phosphate, in the transglycosylation reaction.