Biosynthesis Of Purine Alkaloids Research Articles

CAFFEINE BIOSYNTHESIS AND PURINE METABOLISM IN LEAVES OF MASCAROCOFFEA SPECIES

Caffeine, a purine alkaloid, was not detected in leaves of two Mascarocoffea species, Coffea millotii and Coffea perrier. Trigonelline, a pyridine alkaloid, occurred in these species, but the levels (3−4 μmol/g fresh weight) were much lower than that of Robusta coffee (Coffea canephora) (36 μmol/g fresh weight). Feeding experiments with [8-14C]adenine indicated that purine alkaloid biosynthesis was terminated at 7-methylxanthine formation and as a consequence theobromine and caffeine were not produced in Coffea millotii and Coffea perrier. The adenine salvage activity was lower but its degradation activity was higher in leaves of these Mascarocoffea species than those in Coffea canephora. The metabolic fate of the purine nucleosides, [8-14C]inosine, [8-14C]guanosine and [8-14C]xanthosine was investigated in leaves of Coffea millotii. The biosynthesis of 7-methylxanthine, but not theobromine or caffeine, from these precursors was detected. Large amounts of these purine nucleosides were catabolized via allantoin. Limited amounts of [8-14C]inosine and [8-14C]guanosine were salvaged and utilized for RNA synthesis, however, no [8-14C]xanthosine salvage was observed. Little or no 14C-metabolites were observed when [8-14C]theobromine and [8-14C]caffeine were applied to leaf disks of Coffea millotii. From the results obtained in this study, possible metabolic pathways of purines in Mascarocoffea species are discussed.

Profiles of Purine Metabolism and Purine Alkaloid Biosynthesis in Schima and Eurya Plants

Profiles of Purine Metabolism and Purine Alkaloid Biosynthesis in Schima and Eurya Plants

Comparison of catechins and purine alkaloids in albino and normal green tea cultivars (Camellia sinensis L.) by HPLC

The variations in the contents of catechins and purine alkaloids in relation to the chlorophyll contents in albino tea cultivar ‘Anji baicha’ and normal cultivar ‘Longjing 43’, grown in Anji and Guilin, were determined. The results showed that, compared to ‘Anji baicha’, ‘Longjing 43’ had significantly higher (−)-epigallocatechin (EGC), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), theobromine and caffeine contents in Anji, and significantly higher (+)-catechin (C), ECG and caffeine contents in Guilin. ‘Anji baicha’ only had C content significantly higher than ‘Longjing 43’ in Anji. Correlation analysis indicated that C, EGC and (−)-epigallocatechin gallate (EGCG) were significantly and negatively correlated with chlorophyll contents, suggesting that chlorophylls might play vital roles in the regulation of catechin biosynthesis. Moreover, no significant correlation was observed between purine alkaloids and chlorophylls, indicating that the biosynthesis of purine alkaloids might be affected by genotypic factors other than chlorophyll biosynthesis.

Profiles of Purine Metabolism in Leaves and Roots of Camellia sinensis Seedlings

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.

Biosynthesis of purine alkaloids underlying the diversity of N-methyltransferase

Biosynthesis of purine alkaloids underlying the diversity of N-methyltransferase

Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes

In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8- 14C]adenine was investigated, together with the overall metabolism of [8- 14C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8- 14C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO 2) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8- 14C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g −1 fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.

Distribution and biosynthesis of caffeine in plants.

Methylxanthines and methyluric acids are secondary metabolites derived from purine nucleotides and are known collectively as purine alkaloids. The present review begins by summarizing the distribution of these compounds in the plant kingdom, and then provides an up-to-date account of the biosynthesis of purine alkaloids. The diversity of biosynthesis and accumulation between species and between tissues of different age is considered. We also discuss the physiological function of these purine alkaloids in plants and the biotechnology for creating caffeine-free plants.

Distribution, biosynthesis and function of purine and pyridine alkaloids in Coffea arabica seedlings

Endogenous levels of purine and pyridine alkaloids were studied in different parts of 6-month-old Coffea arabica seedlings. In seedlings, caffeine was distributed mainly in leaves and cotyledons at concentrations varying from 43 to 104 μmol g −1 dry weight. Essentially no caffeine was detected in roots or in older brown parts of shoots. In contrast, trigonelline was present in all parts of the seedlings. The concentration of trigonelline was highest in the upper part of the stems, including buds, which consist of young cells (180 μmol g −1 dry weight), and was lowest in roots (25 μmol g −1 dry weight). The trigonelline concentration in leaves was 60–80 μmol g −1 dry weight, and the concentration in young leaves was higher than in older leaves. Purine alkaloid biosynthesis was estimated from the incorporation of radioactivity from [8- 14 C] adenosine into purine alkaloids. Theobromine and caffeine were synthesized only in young leaves and young shoots including buds, but no biosynthetic activity was found in roots or aged cotyledons. Biosynthetic activity of trigonelline was estimated from the conversion of exogenously supplied [carboxyl- 14 C] nicotinic acid to trigonelline. Trigonelline synthesis was found in all parts of the coffee seedlings. Metabolic fate studies indicated that large fractions of the radioactivity from [carboxyl- 14 C] nicotinic acid (59% in leaves, 53% in cotyledons, 36% in stems and 29% in roots) were incorporated into trigonelline during a 4 h incubation period. Radioactivity was also found in NAD(P), NMN and nicotinamide. Only trace amounts of 14 CO 2 from [carboxyl- 14 C] nicotinic acid were detected. These results suggest that caffeine accumulation is specific to above ground parts (leaves, cotyledons and shoots) of the seedlings and that biosynthesis is performed only in very young tissues, whereas trigonelline is distributed in all parts of coffee seedlings and biosynthetic activity is present even in mature parts. The differing roles of these two alkaloids are discussed.

Purine Alkaloid Biosynthesis in Young Leaves of Camellia sinensis in Light and Darkness

C] adenine to young leaves of tea (Camellia sinensis L.). Light did not have any significant influence on the levels of radioactivity associated with the purine alkaloids. The long-term effects of light on caffeine production were studied using young shoots from plants that were maintained in almost complete darkness (1% full sunlight) by being covered with black lawn cloth. In the control shoots of the naturally-grown plants there were net increases in the total purine alkaloid contents of 2,430 nmoles shoot−1, while in shoots that had been in darkness for 7 days much lower increases, 564 nmoles shoot−1, were observed. Caffeine synthase (CS) activity was 332±55 pkat shoot−1 in light which is ca. 40% higher than the 237±37 pkat shoot−1 in plants kept darkness for 7-days. However, a similar pattern of the metabolism of [8-14C] adenine was observed in naturally-grown and dark-grown shoots. These findings indicate light is not essential for the biosynthesis of caffeine in young tea shoots. The lower net formation of caffeine in shoots maintained in darkness is an indirect consequence of the reduced growth rate of the young shoots in the absence of light.

Purification and characterization of caffeine synthase from tea leaves.

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.

Light irradiation causes physiological and metabolic changes for purine alkaloid production by a Coffea arabica cell suspension culture

Light irradiation not only enhanced purine alkaloid (caffeine and theobromine) production by a Coffea arabica cell suspension culture, but also caused physiological changes in cell growth, and sugar and oxygen uptake rates. Both sugar and oxygen uptake rates showed maxima between 7 and 10 W/m 2 of light intensity, where the specific production rate reached the highest, although the specific cell growth rate was reduced by more than 50%. Light acts as a stress for enhanced production. The activities of enzymes related to purine alkaloid production were induced by light after a lag-time of 1 day. Light is an inducer for these enzymes. However, the de novo synthesis of purine alkaloids required at least 6 days of light irradiation. This discrepancy was cuased by the insufficient supply of purine rings, the precursors for biosynthesis of purine alkaloids.

Are xanthosine and 7-methylxanthosine caffeine precursors?

Although caffeine biosynthesis has been reported to start with the methylation of xanthosine to yield 7-methylxanthosine, we failed to demonstrate in suspension-cultured cells of coffee the presence of 7-methylxanthosine, even after considerable stimulation of overall purine alkaloid (PA) formation by means of ethephon or/and adenine. Hence, in order to increase sensitivity and to study incorporation kinetics, stimulation was combined in the present work with radioactive labelling; [ 14C] adenine or [methyl- 14C] methionine was fed to cells with PA synthesis enhanced either by 5 mM ethephon or by 1 mM adenine. Ethephon treatment resulted in a high (50 to 80% after 24 hr) incorporation of adenine radioactivity into PA, whereas pre-incubation with ‘cold’ adenine known to increase long-term PA formation strongly reduced that incorporation in favour of labelling xanthosine and related nucleosides. Neither ethephon nor adenine treatment led to a distinct incorporation of [ 14C]adenine or [methyl- 14C]methionine into 7-methylxanthosine. Traces of [ 14C]7-methylxanthosine (less than 1%) could only be detected in cells pre-incubated with 1 mM adenine and thus exhibiting ‘artificially’ enlarged endogenous xanthosine pools either strongly labelled (after [ 14C]adenine-feeding) or unlabelled (after [methyl- 14C]methionine-feeding). Kinetic analyses upon labelling with [ 14C]adenine revealed that, based on the specific radioactivity, xanthosine must be excluded as a precursor in caffeine biosynthesis, unless we consider separate pools of xanthosine, one being a collecting pool, the other the PA biosynthesis pool. Therefore, if we adhere to the xanthosine hypothesis, we should postulate metabolic channelling of the early events in PA biosynthesis, including formation of xanthosine, its methylation to 7-methylxanthosine, and finally enzymatic hydrolysis to 7-methylxanthine. However, currently there is no argument against the possibility of the first methylation taking place at the nucleotide level.

Purine metabolism and the biosynthesis of caffeine in maté leaves

Purine metabolism and the biosynthesis of purine alkaloids in leaves of maté plants were investigated by administering [8 −14C]adenine,[8 −14C]guanosine and [8 −14C]hypoxanthine to maté plants of different ages. Each precursor was incorporated into theobromine and caffeine in young leaves. No biosynthesis of these purine alkaloids took place in mature dark-green leaves. Pulse-chase experiments and the inhibitory effects of coformycin suggest that the biosynthesis of caffeine from adenine nucleotides is initiated by the reaction catalysed by AMP deaminase. The conventional pathway for the degradation of purines via allantoin and allantoic acid appeared to be operative in both young and mature leaves. No significant degradation of caffeine was detected in the tracer experiments. The levels of adenine nucleotides in young maté leaves were about five times higher than those of guanine nucleotides. Other possible pathways of purine metabolism in maté leaves are discussed.

Biosynthesis of Caffeine in Flower Buds of Camellia sinensis

The biosynthesis of purine alkaloids in flower buds of tea plants was investigated. More than 25% of total radioactivity of [8-14C]adenine taken up by stamens isolated from tea flower buds was found to have been incorporated into purine alkaloids, namely, theobromine and caffeine, 24 h after administration of the labelled compound. Pulse-chase experiments indicated that [8-14C]adenine taken up by the stamens was converted to adenine nucleotides and subsequently incorporated into theobromine and caffeine. Since 5 μM coformycin, an inhibitor of AMP deaminase, inhibited the incorporation of radioactivity into the purine alkaloids, synthesis of caffeine from adenine nucleotides seems to be initiated by the reaction of AMP deaminase. Although most of the radioactivity from [8-14C]inosine was recovered as CO2 and ureides, considerable amounts of radioactivity were recovered as purine alkaloids. The incorporation of radioactivity from [8-14C]inosine into the purine alkaloids was not affected by coformycin. The five enzymes involved in synthesis of 5-phosphoribosyl-1-pyrophosphate from glucose were present in the stamens and petals of tea flower buds. From present and previous results, the pathway for the biosynthesis of caffeine from adenine nucleotides in flower buds of tea is discussed.