In co¡ee and tea plants, ca¡eine is synthesized from xanthosine via a pathway that includes three methylation steps. We report the isolation of a bifunctional co¡ee ca¡eine synthase (CCS1) clone from co¡ee endosperm by reverse transcriptionpolymerase chain reaction (RT-PCR) and rapid ampli¢cation 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 ca¡eine synthase (TCS1). Interestingly, CCS1 has dual methylation activity like tea TCS1.
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, adenosineagarose, 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 K m values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 M, 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.
Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) have been found in more than 60 subtropical plant species (Suzuki et al., 1992). Many plants used for nonalcoholic beverages, such as tea (Camellia sinensis L.), coffee (Coffea arabica L.), cocoa (Theobroma cacao), and maté (Ilex paraguariensis), contain these purine alkaloids (Suzuki and Waller, 1988). The biosynthesis pathway of theobromine and caffeine has been the subject of much study over the years. Although early investigations implied the involvement of nucleic acids as precursors in caffeine biosynthesis (Ogutuga and Northcote, 1970; Suzuki and Takahashi, 1976), more recent investigations with tea and coffee suggest that caffeine is produced from the purine nucleotides AMP, GMP, and/ or IMP and that theobromine is the immediate precursor of caffeine ( Fig. 1) (Negishi et al., 1985(Negishi et al., , 1992Fujimori et al., 1991;Suzuki et al., 1992;Fujimori and Ashihara, 1994). In coffee, young leaves that are not fully expanded have the highest capacity for caffeine biosynthesis (Fujimori and Ashihara, 1994).
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations –citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.