Uracil synthesisvia HCN oligomerization

Uracil is released from HCN oligomers upon acid hydrolysis in concentrations of 0.001% for 1M HCN solutions to 0.005% for 0.1M solutions. This yield is comparable with earlier reported, minor or nonbiological pyrimidines such as 5-hydroxyuracil and orotic acid. This is the first report of uracil itselfvia HCN oligomerization. Data are presented which establish that the observed uracil is not formed by decarboxylation of previously formed orotic acid, butvia acid hydrolysis of at least two other precursors.     

HCN: A plausible source of purines,pyrimidines and amino acids on the primitive earth

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 9.2. Hydrolysis of these oligomers yields 4,5-dihydroxypyrimidine, orotic acid, 5-hydroxyuracil, adenine, 4-aminoimidazole-5-carboxamide and amino acids. These results, together with the earlier data, demonstrate that the three main classes of nitrogen-containing biomolecules, purines, pyrimidines and amino acids may have originated from HCN on the primitive earth. The observation of orotic acid and 4-aminoimidazole-5-carboxyamide suggests that the contemporary biosynthetic pathways for nucleotides may have evolved from the compounds released on hydrolysis of HCN oligomers.     

Chemical evolution XXIX. Pyrimidines from hydrogen cyanide.

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 8-9. Hydrolysis of these oligomers at pH 8.5 or with 6 N HCl yields 4,5-dihydroxypyrimidine, as the most abundant pyrimidine product along with orotic acid and 5-hydroxyuracil. These results, together with the earlier data, demonstrate that the three major nitrogen-containing classes of biomolecules could have originated from HCN on the primitive earth. The observation of the formation of orotic acid and 4-aminoimidazole-5-carboxamide by the hydrolysis of the HCN oligomers suggests that once the initially formed pyrimidines and purines were consumed, those life forms persisted which evolved enzymes for conversion of these intermediates to the pyrimidines and purines present in contemporary RNA.     

Biomolecules from HCN

The mechanism of the condensation of dilute aqueous solutions of HCN and the products formed by these reactions have been investigated. The initial HCN condensation reactions yield3, a compound which is readily oxidized to4. A similar oxidation of5 to6 was also observed. Urea is formed on hydrolysis of4. The oxidation-reduction products formed from HCN may be in part a consequence of the oxidation of3. It has been established by combination GC/MS that the amino acids glycine, diaminosuccinic acid, α-amino-isobutyric acid, aspartic acid, alanine and isoleucine are released on acid hydrolysis of the ‘HCN polymer’. Hydantoin (7), 5,5-dimethylhydantoin (8) and 5-carboxymethyldenehydantoin (10) are also released on acid hydrolysis of the HCN condensation products. The direct conversion of the dicarbonyl derivative, of diaminosuccinic acid to orotic acid via10 at pH 8 has been observed. This conversion suggests a direct route to pyrimidines from HCN.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

Determination of urinary orotic acid and uracil by capillary zone electrophoresis

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperornithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

Orotic acid excretion in some wild-type strains of Escherichia coli K-12.

During rapid growth, the excretion of pyrimidines, predominantly uracil, is a common phenomenon in procaryotes and eucaryotes. In Escherichia coli, some K-12 strains excrete orotic acid and not uracil. This is caused by a mutation in the pyrF gene.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

嘧啶核苷类物质乳清酸产生菌的选育

以谷氨酸棒杆菌JSIM-201菌株为出发菌株,通过紫外线和甲基磺酸乙酯诱变处理,得到了一株尿嘧啶营养缺陷型突变体U-12菌株,能以葡萄糖为碳源,硫酸铵为氮源,在发酵液中积累一种紫外吸收物质。对U-12菌株的发酵液分离提取结晶,经物理、化学分析鉴定,证明是乳清酸物质。发酵液中积累乳清酸8．6g/L。     

Prebiotic adenine synthesis via HCN oligomerization in ice

Adenine is produced (after hydrolysis) when 0.01 M solutions of HCN are adjusted to pH 9.2 with NH4OH and are frozen at -2 degrees C for 60-100 days. The addition of glycolonitrile (the cyanohydrin of formaldehyde) increases the yield of adenine under these conditions by about five-fold. These results confirm and extend an earlier suggestion that purine synthesis on the prebiotic Earth might have occurred in frozen, dilute solutions of HCN.     

Bacteriophage phi W-14-infected Pseudomonas acidovorans synthesizes hydroxymethyldeoxyuridine triphosphate.

The infection of Pseudomonas acidovorans with bacteriophage phi W-14 leads to the gradual disappearance of dTTP from the cells and to the appearance of hydroxymethy dUTP (hmdUTP). Infected-cell contain dUMP hydroxymethylase and activities converting hmdUMP to humdUDP and hmdUTP. Hydroxymethylase appears immediately after infection, reaching a maximum 20 min later. Thymidylate synthase activity decreases to less than 10% of the preinfection level during the initial 40 min after infection. Newly replicated DNA contains 2 to 3% hydroxymethyluracil. Although uracil is released from newly replicated DNA by acid hydrolysis, uracil is not incorporated as such into phi W-14 DNA, and dUTP is not present in the acid-soluble pool of infected cells. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are formed from hydroxymethyluracil at the polynucleotide level and that an intermediate in one or both of these conversions is degraded to uracil by acid hydrolysis. The modification of hydroxymethyluracil is coupled tightly to replication.     

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.     

Pyrimidine metabolism during somatic embryo development in white spruce (Picea glauca)

Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-¹⁴C]orotic acid, an intermediate of thede novo pathway, and with [2-¹⁴C]uridine and [2-¹⁴C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-¹⁴C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as ¹⁴CO₂, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.     

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, diphydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.     

Verwertung von Pyrimidinderivaten durch Hydrogenomonas facilis

Zusammenfassung Nach Behandlung mit 1-Nitroso-3-nitro-1-methylguanidin und nach Anreicherung in einem penicillinhaltigen Medium wurden von Hydrogenomonas facilis 35 Mutanten isoliert, die Uracil nicht mehr als N-Quelle zu nutzen vermochten. Eine Gruppe dieser Mutanten bildete keine Dihydrouracil-Dehydrogenase und verwertete Thymin, Orotsäure und Uracil nicht mehr. Eine zweite Gruppe hatte die Fähigkeit verloren, Dihydrouracil-Hydrase zu bilden und konnte Uracil, Orotsäure, Thymin, Dihydrouracil und Dihydrothymin nicht mehr verwerten. Während des Wachstums mit Cytosin wurde durch die erste Gruppe dieser Mutanten Uracil und durch die zweite Gruppe Dihydrouracil in das Nährmedium ausgeschieden.Die Enzyme Dihydrouracil-Dehydrogenase und Dihydrouracil-Hydrase waren in Zellen, die mit Cytosin, Uracil, Thymin oder Orotsäure angezogen worden waren, mit wesentlich höherer spezifischer Aktivität nachweisbar als in Zellen, die mit Ammoniumchlorid gewachsen waren. Dihydroorotsäure-Dehydrogenase und Dihydroorotsäure-Hydrase waren in den zellfreien Extrakten in keinem Fall nachweisbar. Die Befunde weisen daraufhin, daß Uracil und Thymin bei H. facilis durch eine unspezifische Dehydrogenase und Dihydrouracil und Dihydrothymin durch eine unspezifische Hydrase umgesetzt werden, und daß diese Enzyme in Gegenwart von Uracil, Thymin oder Orotsäure induktiv gebildet werden.

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Utilization of pyrimidine derivatives by Hydrogenomonas facilis II. Degradation of thymine and uracil by wild type and mutants

Summary 35 mutant strains, unable to utilize uracil as a nitrogen source, were isolated from Hydrogenomonas facilis following treatment with 1-nitroso-3-nitro-1-methylguanidine and enrichment in a penicillin containing medium. One group of these mutants lacked dihydrouracil dehydrogenase and did not utilize thymine, orotic acid and uracil. A second group of mutants had lost the ability to form dehydrouracil hydrase and was unable to utilize uracil, orotic acid, thymine, dihydrouracil and dihydrothymine. The first group of these mutants excreted uracil, the second group dihydrouracil into the medium during growth with cytosine.The enzymes dihydrouracil dehydrogenase and dihydrouracil hydrase were present in much higher specific enzyme activities in cells grown with cytosine, uracil, thymine or orotic acid than in ammonia grown cells. Dihydroorotic dehydrogenase and dihydroorotase could not be demonstrated in cell-free extracts. These data indicate that both uracil and thymine are utilized as substrates by a non-specific hydrogenase and that both dihydrouracil and dihydrothymine are utilized by a non-specific hydrase. Both these enzymes are induced in presence of uracil, thymine or orotic acid in cells of Hydrogenomonas facilis.

     

Prebiotic synthesis of orotic acid parallel to the biosynthetic pathway

By heating an aqueous solution of aspartic acid and urea, carbamylaspartic acid is first formed and then the molecule is cyclized to dihydroorotic acid (DHO) with loss of water. Irradiation of an aqueous solution of DHO with a tungsten lamp yields orotic acid by photo-dehydrogenation of the molecule. This pathway of orotic acid formation is quite similar to that of biosynthesis of the molecule.     

Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Isolation and genetic study of Aspergillus nidulans mutants defective in pyrimidine biosynthesis]

8 uridine-requiring pyr mutants were isolated from Aspergillus nidulans under nitrosoguanidine treatment. All the mutants are capable to grow on the medium containing 20 mkg/ml of uridine or cytidine, or 100 mkg/ml of uracil, and they do not utilize thymidine, thymine, cytosine and deoxyuridine. Their ability to grow in the presence of orotic acid demonstrates that the pyrimidine synthesis in all the mutants is blocked at stages preceding the conversion of orotic acid into orotidine monophosphate. All the pyr mutants are of nuclear nature, they are recessive and represent three complementation groups located in the VIII chromosome. Unlike U. maydis mutant, the requirement in pyrimidines does not increase the sensitivity of A. nidulans pyr mutants to UV-irradiation.     

Contribution of enzyme-phosphoribosyl contacts to catalysis by orotidine 5'-phosphate decarboxylase

The crystal structure of the complex formed between recombinant yeast orotidine 5'-phosphate decarboxylase and the competitive inhibitor 6-hydroxyuridine 5'-phosphate reveals the presence of four hydrogen bonds between active site residues Tyr-217 and Arg-235 and the phosphoryl group of this inhibitor. When Tyr-217 and Arg-235 are individually mutated to alanine, values of k(cat)/K(m) are reduced by factors of 3000- and 7300-fold, respectively. In the Y217A/R235A double mutant, activity is reduced more than 10(7)-fold. Experiments with highly enriched [(14)C]orotic acid show that when ribose 5'-phosphate is deleted from substrate orotidine 5'-phosphate, k(cat)/K(m) is reduced by more than 12 orders of magnitude, from 6.3 x 10(7) M(-1) s(-1) for OMP to less than 2.5 x 10(-5) M(-1) s(-1) for orotic acid. Activity toward orotate is not "rescued" by 1 M inorganic phosphate. The K(i) value of ribose 5'-phosphate, representing the part of the natural substrate that is absent in orotic acid, is 8.1 x 10(-5) M. Thus, the effective concentration of the 5'-phosphoribosyl group, in stabilizing the transition state for enzymatic decarboxylation of OMP, is estimated to be >2 x 10(8) M, representing one of the largest connectivity effects that has been reported for an enzyme reaction.