Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi

McElwain, M C; Pollack, Jonathan D.

doi:10.1128/jb.169.8.3647-3653.1987

Cited by 25 publications

(26 citation statements)

References 44 publications

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“…It is known that this organism can form pyrimidine deoxyribonucleotides from ribonucleosides, since deoxycytidine is a precursor for DNA synthesis in vivo (40) and fluorodeoxyuridine is inhibitory to growth (42). Presumably, these deoxynucleosides are activated by a kinase activity, as shown for more familiar organisms (28,35). Further investigation of such pathways will complement what is known about salvage and synthesis pathways in this versatile autotroph.…”

Section: Discussionmentioning

confidence: 99%

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

Worrell

Nagle

1990

J Bacteriol

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The enzymes involved in the purine interconversion pathway of wild-type and purine analog-resistant strains of Methanobacterium thermoautotrophicum Marburg were assayed by radiometric and spectrophotometric methods. Wild-type cells incorporated labeled adenine, guanine, and hypoxanthine, whereas mutant strains varied in their ability to incorporate these bases. Adenine, guanine, hypoxanthine, and xanthine were activated by phosphoribosyltransferase activities present in wild-type cell extracts. Some mutant strains simultaneously lost the ability to convert both guanine and hypoxanthine to the respective nucleotide, suggesting that the same enzyme activates both bases. Adenosine, guanosine, and inosine phosphorylase activities were detected for the conversion of base to nucleoside. Adenine deaminase activity was detected at low levels. Guanine deaniinase activity was not detected. Nucleoside kinase activities for the conversion of adenosine, guanosine, and inosine to the respective nucleotides were detected by a new assay. The nucleotide-interconverting enzymes AMP deaminase, succinyl-AMP synthetase, succinyl-AMP lyase, IMP dehydrogenase, and GMP synthetase were present in extracts; GMP reductase was not detected. The results indicate that this autotrophic methanogen has a complex system for the utilization of exogenous purines.As the terminal organisms in many anaerobic food chains, the methanogens play a key role in biodegradation reactions. These organisms are phylogenetically diverse and metabolically rather limited, being capable of methanogenesis from only a few simple substrates (20, 21). They utilize novel coenzymes, lipids, cell walls, and metabolic reactions (3,20,21,37). Molecular biology studies have revealed aspects of gene organization and regulation (6, 21) that support the inclusion of methanogens in the kingdom Archaebacteria and show their similarity to both eubacteria and eucaryotes. The genetic approach to the study of these organisms has yielded mutants and physiological information, but genetic exchange mechanisms are in the very early stages of development (4, 32, 45).Although great strides have been made in understanding methanogens, little is known about the biosynthesis of nucleotides in these organisms. Purine nucleotides can be synthesized from simple precursors by de novo pathways; however, salvage of preformed bases and nucleosides can provide an energy-efficient alternative. Salvage pathways have been found in eucaryotes (1, 13, 16) and eubacteria (12, 43) and appear to be present in archaebacteria (5,23,33,42). Knowledge of purine salvage pathways in methanogens is important because it increases our knowledge of methanogen metabolism, which can be applied to the selection and use of radioactive compounds for labeling nucleic acids and their precursors. It provides insight and understanding of the mechanism of action of purine analogs and allows for the isolation of purine analog-resistant mutants blocked in various steps of the salvage pathways. Because methanogens are resistant to...

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Section: Discussionmentioning

confidence: 99%

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

Worrell

Nagle

1990

J Bacteriol

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“…However, the methanococcal activities were comparable to those found in extracts of Methanobacterium thermoautotrophicum and members of the class Mollicutes (15,29).…”

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confidence: 60%

“…These levels were approximately 2 orders of magnitude lower than those found in the members of Mollicutes, organisms that have an absolute requirement for nucleobases for growth (15). Therefore, it is unlikely that these enzymes are a major route of nucleobase incorporation in M. voltae.…”

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confidence: 79%

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae

1996

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Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min ؊1 mg of protein ؊1 and had an apparent K m for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min ؊1 mg of protein ؊1. Hypoxanthine PRTase activity was present at 0.85 nmol min ؊1 mg of protein ؊1 with an apparent K m for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min ؊1 mg of protein ؊1. PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.De novo synthesis of purine nucleotides requires a minimum of 10 enzymes for the production of IMP, the major intermediate of purine metabolism (6). The salvage of nucleobases and nucleosides provides an energy-saving alternative. Salvage pathways have been identified in eubacteria, eucaryotes, and archaebacteria and comprise a collection of enzymes capable of converting preformed nucleobases and nucleosides to AMP, GMP, IMP, and XMP (5,7,10,13,17,19,21,23,29). These enzymes enable organisms to use exogenous bases liberated from lysed cells or recycle those produced by the breakdown of unstable RNA. A key enzyme in the pathway is phosphoribosyltransferase (PRTase). This enzyme is responsible for the conversion of bases to nucleotide 5Ј-monophosphates by use of 5-phosphorylribose-1-pyrophosphate (PPRP) as the phosphopentose donor. Bases can also be salvaged by nucleoside phosphorylases by use of ribose-1-phosphate as the phosphopentose donor. However, in Escherichia coli and Salmonella typhimurium, this mechanism is a minor source of nucleotides (20).Mutants resistant to nucleobase analogs have been isolated from several archaebacteria (2,12,16,29). As with eubacteria and eucaryotes, these mutants have lost the ability to incorporate certain exogenous bases, and this loss is almost always accompanied by a loss of the corresponding PRTase ac...

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“…In these experiments, the concentration of radioactive adenine was increased from our standard reaction concentration to 200 p.M, while the amount of R-1-P, dR-1-P, or X-1-P was varied over a range of 0.25 to 10.0 mM. Samples were assayed as we have described (11). To determine the Hill or interaction coefficient (n), we also replotted the data for each series of pentose reactions as a Hill plot (4).…”

Section: Methodsmentioning

confidence: 99%

“…PNP activity was assayed essentially as described previously (11). Reaction mixtures, 0.1 ml, for the nucleobase to nucleoside conversion contained 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4, 2 mM MgCl2, [8- (17).…”

Section: Methodsmentioning

confidence: 99%

Acholeplasma laidlawii B-PG9 adenine-specific purine nucleoside phosphorylase that accepts ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate

1988

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An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-l-phosphate, deoxyribose-1-phosphate, or xylose-l-phosphate as the pentose donor. Mg2e and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (R.P 0.57) corresponded to all enzyme activity. The Kms for the partially pwrified PNP with ribose-l-phosphate, deoxyribose-l-phosphate, and xylose-l-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding V11,,s were 12.5, 14.3, and 12.0 FIM min-', respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC 2.4.2

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Synthesis of deoxyribomononucleotides in Mollicutes: dependence on deoxyribose-1-phosphate and PPi

Cited by 25 publications

References 44 publications

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

Genetic and physiological characterization of the purine salvage pathway in the archaebacterium Methanobacterium thermoautotrophicum Marburg

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae

Acholeplasma laidlawii B-PG9 adenine-specific purine nucleoside phosphorylase that accepts ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate

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