Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine

Abstract

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.

Convergent evolution of caffeine in plants by co-option of exapted ancestral enzymes

“…For more than 30 y, published studies have indicated that caffeine is produced via a single canonical pathway in plants (44,45 Caffeine biosynthetic network has 12 potential paths. The only path characterized from plants is shown by solid black arrows and involves sequential methylation of xanthosine at N-7, 7-methylxanthine at N-3, and theobromine at N-1 of the heterocyclic ring.…”

Further investigation of the biosynthesis of caffeine in tea plants (Camellia sinensis L.). Methylation of transfer ribonucleic acid by tea leaf extracts

“…the origin of the purine ring in caffeine, until these methylating systems are demonstrated in vitro. In contrast, assuming that, by pathways analogous to those demonstrated for caffeine biosynthesis by Ogutuga & Northcote (1970), theophylline is synthesized from nucleic acids, it is possible that theophylline is produced from 1-methyladenylic acid, formed as a result of nucleic acid methylation and subsequent degradation, via 1-methylxanthine (Suzuki & Takahashi, 1975).…”

Conversion of Xanthosine into Caffeine in Tea Plants

“…The 7-methylxanthosine found in this experiment was also identified after hydrolyzing to 7-methylxanthine, since the definite result for determination of the position of methyl group at the xanthine ring can be obtained by this method. The hydrolysis product matched 7-methylxanthine (Rf 0.40 in solvent (1) and 0.30 in solvent (5)). …”

“…Elution was done stepwise with O.oI, 0.05, 0.1, 0.2, and O.4M triethylammonium formate buffer (PH 3.8). The radioactive fraction corresponding to 7-methylxanthosine, which was eluted with 0.1 M buffer, was treated with 1 N HCl at 100°C for 1 hr and identified by co-chromatography with authentic methylated xanthines in solvent (1) and tertBuOH-methyl ethyl ketone-H20-28% NH40H (4: 3: 2: 1), solvent (5).…”