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Gert Dandanell - One of the best experts on this subject based on the ideXlab platform.
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Xanthosine utilization in salmonella enterica serovar typhimurium is recovered by a single aspartate to glycine substitution in Xanthosine phosphorylase
Journal of Bacteriology, 2006Co-Authors: Michael Hansen, Jesper Tranekjaer Jorgensen, Gert DandanellAbstract:xapABR from Salmonella enterica was analyzed and compared with the corresponding Escherichia coli genes. xapB and xapR, but not xapA, encode functional proteins. An S. enterica XapA(Asp72Gly) mutant that restores the phosphorolytic activity was selected. The purified mutant enzyme has different kinetic constants than the E. coli enzyme but similar substrate specificity.
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purification and characterization of rihc a Xanthosine inosine uridine adenosine preferring hydrolase from salmonella enterica serovar typhimurium
Biochimica et Biophysica Acta, 2005Co-Authors: Michael Hansen, Gert DandanellAbstract:Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes Xanthosine by Xanthosine phosphorylase (xapA), Salmonella cannot grow on Xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze Xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k(cat)/Km=1 to 4 x 10(4) M(-1)s(-1)). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k(cat)/Km=0.7 to 1.2 x 10(3) M(-1)s(-1)) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and Xanthosine and the hydrolytic capacity (k(cat)/Km) is 5-fold higher for Xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of Xanthosine.
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specificity and topology of the escherichia coli Xanthosine permease a representative of the nhs subfamily of the major facilitator superfamily
Journal of Bacteriology, 2001Co-Authors: Morten H H Norholm, Gert DandanellAbstract:The specificity of XapB permease was compared with that of the known nucleoside transporters NupG and NupC. XapB-mediated Xanthosine uptake is abolished by 2,4-dinitrophenol and exhibits saturation kinetics with an apparent Km of 136 μM. A 12-transmembrane-segment model was confirmed by translational fusions to alkaline phosphatase and the α fragment of β-galactosidase.
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identification and characterization of genes xapa xapb and xapr involved in Xanthosine catabolism in escherichia coli
Journal of Bacteriology, 1995Co-Authors: C Seeger, C Poulsen, Gert DandanellAbstract:We have characterized four genes from the 52-min region on the Escherichia coli linkage map. Three of these genes are directly involved in the metabolism of Xanthosine, whereas the function of the fourth gene is unknown. One of the genes (xapA) encodes Xanthosine phosphorylase. The second gene, named xapB, encodes a polypeptide that shows strong similarity to the nucleoside transport protein NupG. The genes xapA and xapB are located clockwise of a gene identified as xapR, which encodes a positive regulator belonging to the LysR family and is required for the expression of xapA and xapB. The genes xapA and xapB form an operon, and their expression was strictly dependent on the presence of both the XapR protein and the inducer Xanthosine. Expression of the xapR gene is constitutive and not autoregulated, unlike the case for many other LysR family proteins. In minicells, the XapB polypeptide was found primarily in the membrane fraction, indicating that XapB is a transport protein like NupG and is involved in the transport of Xanthosine.
Hiroshi Ashihara - One of the best experts on this subject based on the ideXlab platform.
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Metabolism of alkaloids in coffee plants
2013Co-Authors: Hiroshi AshiharaAbstract:Coffee beans contain two types of alkaloids, caffeine and trigonelline, as major components. This review describes the distribution and metabolism of these compounds. Caffeine is synthesised from Xanthosine derived from purine nucleotides. The major biosynthetic route is Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine → caffeine. Degradation activity of caffeine in coffee plants is very low, but catabolism of theophylline is always present. Theophylline is converted to xanthine, and then enters the conventional purine degradation pathway. A recent development in caffeine research is the successful cloning of genes of N-methyltransferases and characterization of recombinant proteins of these genes. Possible biotechnological applications are discussed briefly. Trigonelline (N-methylnicotinic acid) is synthesised from nicotinic acid derived from nicotinamide adenine nucleotides. Nicotinate N-methyltransferase (trigonelline synthase) activity was detected in coffee plants, but purification of this enzyme or cloning of the genes of this N-methyltransferase has not yet been reported. The degradation activity of trigonelline in coffee plants is extremely low. Key words: Coffea, caffeine, purine alkaloids, pyridine alkaloids, theobromine, trigonelline. Metabolismo de alcalóides em plantas de café: Sementes de café possuem dois tipos de alcalóides, cafeína e trigonelina, como principais componentes. Esta revisão descreve a distribuição e metabolismo desses compostos. Cafeína é sintetizada a partir da xantosina derivada de nucleotídeos purínicos. A principal rota biossintética é xantosina → 7-metilxantosina → 7-metilxantina → teobromina → cafeína. A atividade de degradação de cafeína em café é muito baixa, mas o catabolismo de teofilina está sempr
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Biosynthesis and Catabolism of Purine Alkaloids
New Light on Alkaloid Biosynthesis and Future Prospects, 2013Co-Authors: Hiroshi Ashihara, Takao Yokota, Alan CrozierAbstract:A limited number of plant species accumulate purine alkaloids, such as caffeine and theobromine, which are synthesized from Xanthosine, a catabolite of purine nucleotides. The main biosynthetic pathway is a sequence consisting of Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine → caffeine. This review summarizes the occurrence of purine alkaloids in the plant kingdom, the caffeine biosynthesis routes from purine precursors, the enzymes and genes of N-methyltransferases, key enzymes of caffeine biosynthesis, caffeine catabolism and the possible ecological role of caffeine. Finally, we introduce transgenic plants in which caffeine production is either suppressed or induced by the introduction of caffeine encoded genes. Such plants have the potential to be used for the production of decaffeinated coffee and tea or as natural pesticides in agriculturally important crops.
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Xanthosine metabolism in plants: Metabolic fate of exogenously supplied 14C-labelled Xanthosine and xanthine in intact mungbean seedlings
Phytochemistry Letters, 2012Co-Authors: Hiroshi AshiharaAbstract:Abstract Xanthosine is a catabolite of purine nucleotides. Our studies using excised tissues of various plant species indicate that Xanthosine salvage is negligible and that Xanthosine is catabolised predominantly via xanthine. A recent report using intact Arabidopsis thaliana seedlings (Riegler et al., 2011. New Phytol. 191, 349–359) showed that significant amounts of Xanthosine were utilised for RNA synthesis. We report here similar, more detailed 14C-feeding experiments of Xanthosine and xanthine using intact mungbean seedlings. Less than 3% of radioactivity from [8-14C]Xanthosine and 1% from [8-14C]xanthine was incorporated into the RNA fraction; the rest of the radioactivity was incorporated into purine catabolites, including ureides, urea and CO2. Allopurinol, which is a xanthine oxidoreductase inhibitor, markedly inhibited purine catabolism, and radioactivity from these two precursors was retained in xanthine. Even then, no significant salvage of Xanthosine and xanthine was observed. Rapid catabolism and slow salvage of Xanthosine and xanthine appear to be inherent properties of many plant species.
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profiles of purine metabolism in leaves and roots of camellia sinensis seedlings
Plant and Cell Physiology, 2010Co-Authors: Weiwei Deng, Hiroshi AshiharaAbstract:To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that Xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than Xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.
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Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves
Plant Physiology and Biochemistry, 2003Co-Authors: Yoko Koyama, Misako Kato, Yoshihisa Tomoda, Hiroshi AshiharaAbstract:Abstract We examined the purine alkaloid content and purine metabolism in cacao ( Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g –1 fresh weight), followed by caffeine (0.75 μmol g –1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, 14 C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO 2 ) and to purine alkaloids (3- and 7-methylxanthine, 7-methylXanthosine and theobromine). In contrast, 14 C-labelled xanthine and Xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8- 14 C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-Xanthosine monophosphate → Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from 14 C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8- 14 C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8- 14 C]Theobromine was degraded to CO 2 via 3-methylxanthine, xanthine and allantoic acid. [8- 14 C]Caffeine was catabolised to CO 2 via theophylline (1,3-dimethylxanthine) or theobromine.
Leroy B Townsend - One of the best experts on this subject based on the ideXlab platform.
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synthetic approaches to the guanosine and Xanthosine analogues 5 amino 3 beta d ribofuranosylpyrazolo 3 4 e 1 3 oxazin 7 one and 3 beta d ribofuranosylpyrazolo 3 4 e 1 3 oxazine 5 7 dione and studies of their antitumor potential
Journal of Medicinal Chemistry, 1991Co-Authors: Ruiming Zou, Vladimir G Beylin, Michael P Groziak, Linda L Wotring, Leroy B TownsendAbstract:5-Amino-3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazin-7-o ne has been synthesized via cyclization of the appropriately protected pyrazofurin derivatives and subsequent transformations of the heterocyclic moiety. This guanosine analogue was marginally cytotoxic to L1210 cells in vitro. The Xanthosine analogue 3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazine-5,7-dione was also synthesized, and was found to be highly cytotoxic. It appeared to act as a prodrug of pyrazofurin.
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nucleosides 3 reactions of aica riboside with isothiocyanates a convenient synthesis of isoguanosine and Xanthosine derivatives
Journal of Organic Chemistry, 1991Co-Authors: Ji Wang Chern, Gwo Sen Lin, Chien Shu Chen, Leroy B TownsendAbstract:Cyclodesulfurative annulations of 5-[1-(3-substituted-thioureido)]-1-(β―D-ribofuranosyl)imidazole-4-carboxamides with N,N'-dicyclohexylcarbodiimide are described. Isoguanosine and several 1-substituted isoguanosines are readily prepared by a ring closure of the appropriate resulting 5-(3-substituted-1-ureido)-1-(β-D-ribofuranosyl)imidazole-4-carbonitriles. Treatment of the appropriate 1-substituted isoguanosines under basic conditions has furnished a 1-substituted Xanthosine
Misako Kato - One of the best experts on this subject based on the ideXlab platform.
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Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves
Plant Physiology and Biochemistry, 2003Co-Authors: Yoko Koyama, Misako Kato, Yoshihisa Tomoda, Hiroshi AshiharaAbstract:Abstract We examined the purine alkaloid content and purine metabolism in cacao ( Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g –1 fresh weight), followed by caffeine (0.75 μmol g –1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, 14 C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO 2 ) and to purine alkaloids (3- and 7-methylxanthine, 7-methylXanthosine and theobromine). In contrast, 14 C-labelled xanthine and Xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8- 14 C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-Xanthosine monophosphate → Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from 14 C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8- 14 C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8- 14 C]Theobromine was degraded to CO 2 via 3-methylxanthine, xanthine and allantoic acid. [8- 14 C]Caffeine was catabolised to CO 2 via theophylline (1,3-dimethylxanthine) or theobromine.
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the first committed step reaction of caffeine biosynthesis 7 methylXanthosine synthase is closely homologous to caffeine synthases in coffee coffea arabica l 1
FEBS Letters, 2003Co-Authors: Kouichi Mizuno, Misako Kato, Fumi Irino, Naho Yoneyama, Tatsuhito Fujimura, Hiroshi AshiharaAbstract:In coffee and tea plants, caffeine is synthesized from Xanthosine via a pathway that has three methylation steps. We identified and characterized the gene encoding the enzyme for the first methylation step of caffeine biosynthesis. The full-length cDNA of coffee tentative caffeine synthase 1, CtCS1, previously isolated by the rapid amplification of cDNA ends was translated with an Escherichia coli expression system and the resultant recombinant protein was purified using Ni-NTA column. The protein renamed CmXRS1 has 7-methylxanthine synthase (Xanthosine:S-adenosyl-L-methionine methyltransferase) activity. CmXRS1 was specific for Xanthosine and Xanthosine 5'-monophosphate (XMP) could not be used as a substrate. The K(m) value for Xanthosine was 73.7 microM. CmXRS1 is homologous to coffee genes encoding enzymes for the second and third methylation steps of caffeine biosynthesis.
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isolation of a new dual functional caffeine synthase gene encoding an enzyme for the conversion of 7 methylxanthine to caffeine from coffee coffea arabica l 1
FEBS Letters, 2003Co-Authors: Kouichi Mizuno, Hiroshi Ashihara, Misako Kato, Naho Yoneyama, Akira Okuda, Hiromi Tanaka, Tatsuhito FujimuraAbstract:In coffee and tea plants, caffeine is synthesized from Xanthosine via a pathway that includes three methylation steps. We report the isolation of a bifunctional coffee caffeine synthase (CCS1) clone from coffee endosperm by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique using previously reported sequence information for theobromine synthases (CTSs). The predicted amino acid sequences of CCS1 are more than 80% identical to CTSs and are about 40% similar to those of tea caffeine synthase (TCS1). Interestingly, CCS1 has dual methylation activity like tea TCS1.
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caffeine biosynthesis in young leaves of camellia sinensis in vitro studies on n methyltransferase activity involved in the conversion of Xanthosine to caffeine
Physiologia Plantarum, 1996Co-Authors: Misako Kato, Tomomi Kanehara, Hisayo Shimizu, Takeo Suzuki, Fiona M Gillies, Alan Crozier, Hiroshi AshiharaAbstract:The aim of this study was to investigate the S-adenosylmethionine dependent N-methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea (Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When Xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of Xanthosine to caffeine. The apparent M r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a Xanthosine → 7-methylXanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.
Michael Hansen - One of the best experts on this subject based on the ideXlab platform.
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Xanthosine utilization in salmonella enterica serovar typhimurium is recovered by a single aspartate to glycine substitution in Xanthosine phosphorylase
Journal of Bacteriology, 2006Co-Authors: Michael Hansen, Jesper Tranekjaer Jorgensen, Gert DandanellAbstract:xapABR from Salmonella enterica was analyzed and compared with the corresponding Escherichia coli genes. xapB and xapR, but not xapA, encode functional proteins. An S. enterica XapA(Asp72Gly) mutant that restores the phosphorolytic activity was selected. The purified mutant enzyme has different kinetic constants than the E. coli enzyme but similar substrate specificity.
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purification and characterization of rihc a Xanthosine inosine uridine adenosine preferring hydrolase from salmonella enterica serovar typhimurium
Biochimica et Biophysica Acta, 2005Co-Authors: Michael Hansen, Gert DandanellAbstract:Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes Xanthosine by Xanthosine phosphorylase (xapA), Salmonella cannot grow on Xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze Xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k(cat)/Km=1 to 4 x 10(4) M(-1)s(-1)). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k(cat)/Km=0.7 to 1.2 x 10(3) M(-1)s(-1)) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and Xanthosine and the hydrolytic capacity (k(cat)/Km) is 5-fold higher for Xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of Xanthosine.
