Bacterial Synthesis of Nucleotides

The synthesis of inosinic acid from inosine and p-nitrophenylphosphate by the partially purified enzyme, nucleoside phosphotransferase, prepared from Escherichia coli (B-25) is described.

The results presented in this paper represent that the nucleotide, inosinic acid, synthesized by the nucleoside phosphotransferase of E. coli, used as an example of bacterial enzymes, is not always 5′-isomer and that most of inosinic acid synthesized are 3′(& 2′)-isomer, together with a small amount of 5′-isomer. It was pointed out that cupric ion accelerated both the synthesis of inosinic acid and the liberation of p-nitrophenol, and that the nucleoside phosphotransferase and the phosphatase may be different from each other.     

Nucleotide,nucleoside and base nutritional requirements of the mosquito Culex pipiens

Nucleic acid subcomponents needed to satisfy the dietary nucleic acid requirement of Culex pipiens were studied in growth experiments using synthetic media in which nucleosides, bases and alternative nucleotides were variously substituted in mixtures of 3 nucleotides (adenylic acid, thymidylic acid, and either cytidylic or uridylic acid) previously shown to be adequate replacements for whole nucleic acid. Any or all 3 nucleotides could be replaced by corresponding nucleosides without adverse effect, except that adenosine substitution moderately delayed pupation. All base substitutions were unsatisfactory: substitution of thymine for thymidylic acid allowed development to the adult stage but at a greatly reduced rate; single substitution of adenine, cytosine or uracil for the corresponding nucleotides allowed scarcely more development than in the total absence of nucleic acid derivatives. Inosinic acid or inosine were adequate substitutes for adenylic acid, but orotic acid or orotidine were ineffective in place of the pyrimidine ribonucleotides, cytidylic or uridylic acids. Deoxyadenylic acid could take the place of adenylic acid, though inefficiently, but deoxycytidylic and deoxyuridylic acids were very poor replacements for the corresponding ribonucleotides. The minimal required nucleic acid derivatives thus appear to be a purine ribonucleotide (adenylic or inosinic acids), a pyrimidine ribonucleoside (either uridine or cytidine), and the pyrimidine deoxyribonucleoside, thymidine.     

A conformational basis for the selective action of ara-adenine.

The glycosidic “high anti” conformation is postulated to be the conformation required by the enzymes adenosine kinase and inosine phosphorylase. Purine analogs that are stable in this conformation are either effective substrates or inhibitors of these enzymes. Ara-adenine is shown to be highly unstable in the high anti conformation. The inactivity of ara-adenine as a substrate for both adenosine kinase and inosine phosphorylase is attributed to its inability to assume the high anti conformation specified by these enzymes. That adenosine itself has a local minima in the high anti conformation, as does inosine and guanosine, is required by its ability to inhibit the synthesis of uridylic acid.The minimal cytotoxic properties of ara-adenine is a consequence of its failure, in normal cells, to be converted to the toxic nucleotide form. The ability of ara-adenine to selectively inhibit DNA viruses means that in DNA virus infected cells the conversion of ara-adenine to ara-AMP is facilitated through a mechanism that does not require a substrate high anti conformation.It is apparent that selective antiviral and anticancer nucleoside analogs may be constructed if their conversion to the toxic nucleotide form is prohibited in normal tissues but allowed in cancer cells or virus infected cells. The basis for the selective effects of ara-adenine is that normal cells require a substrate conformation in which ara-adenine is unstable but that certain neoplastic and viral mechanisms for the conversion of ara-adenine to ara-AMP exist which are able to utilize ara-adenine in its stable syn or anti conformations.     

Adaptation in Lesch-Nyhan cells exposed to aminopterin

Fibroblasts from a particular patient with Lesch-Nyhan syndrome developed full resistance to aminopterin, in association with a progressive increase in ³H-hypoxanthine incorporation. This represented a gradual and generalized adaptation. When aminopterin treatment was stopped, the minimal ³H-hypoxanthine uptake of untreated cells was re-established. The hypoxanthine-guanine phosphoribosyltransferase activity of extracts of these cells was as low as in other Lesch-Nyhan cultures, but aminopterin-treated cells contained more than twice as much activity, suggesting that an increased amount of enzyme caused the adaptation. However, this conlusion can only be tentative in view of the complex and circular nature of the interconversions of hypoxanthine, inosine and inosinic acid in these extracts.     

Adenosine Inhibition of Human Platelet Aggregation by ADP

WE have shown that in human platelet-rich plasma, inosine is a concentration-dependent inhibitor of adenosine incorporation into platelets^1,2 and at high concentrations inosine inhibits adenosine decomposition³. This prompted us to investigate the effect of inosine and other adenosine decomposition products on aggregation of human platelets in vivo by ADP.     

Role of Inosine Monophosphate Oxidoreductase in the Formation of Ureides in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata L. Walp.)

Cell-free extracts from nodules of cowpea (Vigna unguiculata L. (Walp.) cv Caloona:Rhizobium strain CB756) prepared in the presence of 15% (v/v) glycerol showed high rates (30 to 60 nanomoles NAD reduced per minute per gram fresh weight nodule) of inosine monophosphate oxidoreductase (EC 1.2.1.14) activity. The enzyme was labile (half-life of activity less than 3 hours) but could be stabilized for up to 18 hours by inclusion of the substrates NAD and inosine monophosphate in the breaking media. Activity showed a broad pH optimum between 8.5 and 9.5, had an apparent K_m (inosine monophosphate) of 4 and 12 micromolar at pH 7.5 and 9.0, respectively, and was largely (96%) associated with the plant cell cytosol fraction of the nodule.

Metabolism of [8-¹⁴C]inosine monophosphate and [1-¹⁴C]glycine by the cell-free system showed two pathways for purine base production from inosine monophosphate, one via xanthosine monophosphate, xanthosine, and xanthine, the other via inosine and hypoxanthine. The proportion of inosine monophosphate utilized by inosine monophosphate oxidoreductase and the xanthine-based pathway was increased from 30% at 0.5 millimolar to 80% at 0.01 millimolar inosine monophosphate. The data are interpreted to indicate that in vivo inosine monophosphate oxidation rather than dephosphorylation is the predominant metabolic route leading to ureide synthesis and that inosine monophosphate provides the link between de novo purine nucleotide synthesis in the plastid and ureide production in the plant cell cytosol.

     

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots

The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with K_m values of about 160 and 240 micromolar and with V_max values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a K_m of about 700 micromolar and a V_max of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form, the enzyme appears to be fully active, exhibiting lower K_m values to hydrolyze inosine diphosphate. Furthermore, we found that native inosine diphosphatase is inhibited either by ATP or pyrophosphate, whereas inosine diphosphate inhibits the ATPase, but has no significant effect on the pyrophosphatase. It appears that inosine diphosphate is a positive modulator of the inosine diphosphatase, whereas ATP and pyrophosphate act as negative modulators of this enzyme.     

Photoreactivities of 5-Bromouracil-containing RNAs

5-Bromouracil (^BrU) was incorporated into three types of synthetic RNA and the products of the photoirradiated ^BrU-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA^BrUGC)₂ and r(CGAA^BrUUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine (^ketoA) product at the 5′-neighboring nucleotide, such as r(GC^ketoAUGC) and r(CGA^ketoAUUGC), respectively. The photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in Z-form RNA produced the 2′-keto guanosine (^ketoG) product r(CGC^ketoGUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.     

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.

Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K_m value computed for inosine is 65 micromolar.

Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V_max/K_m) towards adenosine is 48 times lower than that towards inosine.

In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The inhibitory effects of 20 compounds including purine analogues on the growth of Micrococcus glutamicus No. 534–348, an inosinic acid-producing adenine-auxotroph, and No. 534, a prototrophic strain, were investigated.

A number of strains resistant to each of 6-merca ptoguanine, 6-thioguanine, 8-azaguanine, mitomycin C and sulfanilamide were induced from strain No. 534–348, and their inosinic acid productivities were compared with their parent strain. Among them, MGR-25, α 6-mercaptoguanine-resistant strain, accumulated more inosinic acid than its parent strain. Furthermore, the strain MGR-25 was differentiated in its morphology, frequency of spontaneous reversion to prototrophic type in adenine-deficient medium, and the effectiveness of hypoxanthine to increase the inosinic acid accumulation.     

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ? phosphate + adenosine (adenosine phosphorylase); adenosine + H₂O → inosine + NH₃ (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.     

Conversion of 5′-inosinic acid to inosine by Streptomyces aureus

The production of inosine by microbial conversion of 5′-inosinic acid (IMP) was investigated. Among the various strains of Streptomyces and Bacillus tested, Streptomyces aureus NCIB 9803 was selected for the microbial conversion process due to its high IMP-degrading activity. A maximum conversion yield of 0.43 (86% of the theoretical value) was obtained when IMP was added to the culture medium at 24 h. Kinetic studies with [8-¹⁴C] IMP showed that the difference from the theoretical values mainly attributable to the uptake of inosine by S. aureus.     

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.     

Adenosine aminohydrolase inhibition in cosolvent--buffer mixtures

Adenosine aminohydrolase from calf intestinal mucosa is sensitive to changes in the cooperative water structure of its environment as induced by the cosolvent dioxane. When dioxane is added to lower the dielectric constant from that of 78 of neat water to about 74, V is approximately halved, competitive inhibition by N⁶-(Δ²-isopentenyl)adenosine is virtually abolished, and competitive inhibition by the product of the reaction, i.e., inosine, is significantly decreased (K_i changes from 0.2 to 0.5 mm inosine). Yet K_m remains unaltered at 40 μm adenosine even to a dielectric constant of 66.Since both N⁶-(Δ²-isopentenyl)adenosine and inosine are competitive inhibitors, they cannot be bound by the enzyme at the same time as adenosine. The fact that substrate binding remains unaltered at dielectric constants where these inhibitors are impotent indicates that binding of these inhibitors by portions of the enzyme not directly involved in substrate binding is important. The degree of alteration of binding with increasing dioxane concentration is different for these two inhibitors, with appreciable inosine binding at mole fractions dioxane where N⁶-(Δ²-isopentenyl)-adenosine binding cannot be demonstrated. Because of this differential effect of dioxane on inosine and N⁶-(Δ²-isopentenyl)adenosine binding, it is apparent that two substances can be competitive inhibitors kinetically and yet be bound differently by an enzyme. Cosolvents may thus be useful probes for the study of enzyme inhibitor interactions. It is proposed that studies of cosolvent effects on enzyme catalysis and substrate and inhibitor binding are capable of revealing the sensitivities of these various sites to alterations in the dielectric constant of the medium and thus may be considered as models for enzyme behavior near cytoplasmic membranes in vivo.     

Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased K_m value for inosine was responsible for the increased productivity.     

The equilibrium of the reaction catalysed by citrate oxaloacetate-lyase

1. A method of preparation and purification of citrate oxaloacetate-lyase (EC 4.1.3.6) from Aerobacter aerogenes is described. 2. The equilibrium of this reaction has been determined at pH 8·4 and 25°. It has been shown that K, i.e. [citrate³⁻]/[oxaloacetate_keto²⁻][acetate ⁻], is 3·08±0·72, but that K_app., i.e. [total citrate]/[total oxaloacetate][total acetate], is markedly affected by the initial concentrations of the reactants and magnesium. 3. The free-energy change during the cleavage of citrate has been calculated and compared with data from other sources. 4. The free energy of hydrolysis of acetyl-CoA has been evaluated from the present data. 5. A detailed knowledge of the interactions of the reactants with metal ions has been shown to be important in the calculation of the equilibrium constant and related thermodynamic functions.     

Roles of α-methyl trans-cyclopropane groups in behavior of mixed mycolic acid monolayers

Mixed Langmuir films of type 1 alpha-(α-) and keto-mycolic acids (MAs) were investigated to understand the roles of α-methyl trans-cyclopropane containing keto-MA in determining the physical and chemical properties of the monolayers. Surface pressure (π) vs. mean molecular area (A) isotherms were measured at constant mole fractions defined as the ratio of the keto-MA molarity to the total molarity of α-MA and keto-MA (X_keto) at 25?°C and 37?°C. A and the elastic modulus (E) of the mixed monolayer were compared for different X_keto at fixed π values. In keto-MA rich monolayers, A values were much larger than values of the combined areas of α-MA and keto-MA, while the E values were close to those of solid keto-MA monolayers. A and E were also plotted against the mole fraction of α-methyl trans-cyclopropane containing keto-MA, which showed that the α-methyl trans-cyclopropane group stabilized the W-form conformation of mycolic acids in monolayers, and rendered them solid state. Furthermore, a comparison of the experimental results and the α-methyl trans-cyclopropane content in cell-wall MAs from various strains indicated that the ratio of trans-cyclopropane content was important in determining the nature of the mixed MA layer.     

A syn conformation for inosine, the wobble nucleoside in some tRNA's

The crystal structure of an orthorhombic form of inosine was determined from three-dimensional X-ray diffraction data. There are two crystallographically independent inosine molecules, both of which assume a $\text{syn}$ conformation that is different from the conformations found in other crystalline forms of inosine. Apparently, inosine has considerable conformational freedom, a property that may be required at the “wobble” position of anticodon triplets.     

The Bacillus cereus GerN and GerT protein homologs have distinct roles in spore germination and outgrowth, respectively

The GerT protein of Bacillus cereus shares 74% amino acid identity with its homolog GerN. The latter is a Na⁺/H⁺-K⁺ antiporter that is required for normal spore germination in inosine. The germination properties of single and double mutants of B. cereus ATCC 10876 reveal that unlike GerN, which is required for all germination responses that involve the GerI germinant receptor, the GerT protein does not have a significant role in germination, although it is required for the residual GerI-mediated inosine germination response of a gerN mutant. In contrast, GerT has a significant role in outgrowth; gerT mutant spores do not outgrow efficiently under alkaline conditions and outgrow more slowly than the wild type in the presence of high NaCl concentrations. The GerT protein in B. cereus therefore contributes to the success of spore outgrowth from the germinated state during alkaline or Na⁺ stress.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.