ReviewCaffeine and related purine alkaloids: Biosynthesis, catabolism, function and genetic engineering
Graphical abstract
The biosynthesis, catabolism and function of caffeine, theobromine and other purine alkaloids are reviewed. Genetic engineering studies which have produced coffee beans with a reduce caffeine content and transgenic caffeine-producing tobacco plants with enhanced disease resistance are discussed.
Introduction
Purine alkaloids are secondary metabolites derived from purine nucleotides (Zulak et al., 2006) that have been found in nearly 100 species in 13 orders of plant kingdom (Ashihara and Crozier, 1999a). Methylxanthines, such as caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine), and methyluric acids are classified as purine alkaloids (Fig. 1). They occur in tea, coffee and a number of other non-alcoholic beverages. Caffeine was isolated from tea and coffee in the early 1820s, but the main biosynthetic and catabolic pathways of caffeine were not fully established until 2000. Highly purified caffeine synthase was obtained from tea leaves after which a gene encoding the enzyme was cloned (Kato et al., 1999, Kato et al., 2000). This facilitated molecular studies on the regulation of caffeine production principally with coffee plants (Ogita et al., 2003). This review describes biosynthesis and catabolism of purine alkaloids, and the genes and molecular structure of N-methyltransferases related to caffeine biosynthesis. In addition, details are included on purine metabolism in a number of plants including Camellia, Coffea, Theobroma and Ilex species. The emerging role of purine alkaloids in planta and metabolic engineering of caffeine are also discussed.
Section snippets
Caffeine biosynthesis
Main caffeine biosynthetic pathway is a four step sequence consisting of three methylation and one nucleosidase reactions (Fig. 2). The xanthine skeleton of caffeine is derived from purine nucleotides. The initial step in caffeine biosynthesis is the methylation of xanthosine by a SAM-dependent N-methyltransferase. In addition to experiments with radiolabelled precursors, substrate specificities of native (Kato et al., 1999) and recombinant N-methyltransferases (Kato et al., 2000, Ogawa et al.,
Genes
Genes encoding enzymes involved in caffeine biosynthesis were successfully isolated from plants of the coffee family by PCR and library screening methods (Ogawa et al., 2001, Uefuji et al., 2003, Mizuno et al., 2003a, Mizuno et al., 2003b). This involved designing degenerated primers based on conserved regions in tea caffeine synthase (AB031280) and Arabidopsis unknown proteins. Small PCR products were then used as the probe to isolate full length cDNA from a library, resulting in the
Catabolism of caffeine
Caffeine is produced in young leaves and immature fruits, and continues to accumulate gradually during the maturation of these organs. However, it is very slowly degraded with the removal of the three methyl groups, resulting in the formation of xanthine. Catabolism of caffeine in coffee leaves was first reported by Kalberer (1965). Since then a number of tracer experiments using 14C-labelled purine alkaloids have been reported (Suzuki and Waller, 1984, Ashihara et al., 1996, Ashihara et al.,
Metabolism of purine alkaloids in individual species
In addition to major pathways of purine alkaloid biosynthesis and catabolism, species dependent minor pathways also exist. In this section, species-specific metabolism and physiological studies will be reviewed.
Role of purine alkaloids in planta
The physiological role of purine alkaloids in planta has until recently remained largely undetermined and it may be that they may be waste end products produced in a limited number of plant species during the course of evolution. Degradation of caffeine is relatively slow even in aged leaves of most species, and it appears not to act as a nitrogen reserve since considerable amounts remain in leaves after abscission. There are two hypotheses concerning the role of caffeine in plants, the
Decaffeinated coffee plants
Identification of genes encoding enzymes for caffeine biosynthesis facilitated engineering the caffeine biosynthetic pathway to either suppress or enhance production. The first approach was to construct transgenic coffee plants with reduced caffeine content by the RNA interference method, in which mRNA of the target gene is selectively degraded by small double-stranded RNA species (Ogita et al., 2003, Ogita et al., 2004, Ogita et al., 2005). The 3′-untranslated region and the coding region of
Summary and perspectives
It is just over seven years since the first report on the cloning of caffeine synthase from tea, the gene encoding the enzyme regulating the final two methylation steps in the caffeine biosynthesis pathway (Kato et al., 2000). Since this pioneering study there has been a veritable explosion of research, most notably by Japanese scientists, that has led to the successful cloning of a number of methyltransferase-encoding genes from coffee, tea and cacao. Much of the wide spread interest in this
Acknowledgements
The authors would like to thank Professor Mike Clifford, University of Surrey, Guildford, UK for the valuable comments he made after reading a draft of the manuscript and Professor Takao Yokota, Teikyo University, Utsunomiya, Japan for his advice on the structures of uric acids in the metabolic pathways illustrated in the text.
Hiroshi Ashihara is Professor of Metabolic Biology at Ochanomizu University in Tokyo. He obtained his PhD from the University of Tokyo in 1975, and did postdoctoral research at the University of Sheffield from 1977 to 1979. His research has focused on primary and secondary metabolism of nucleotides in plants and he has published more than 150 papers on purine, pyrimidine and pyridine nucleotide metabolism. He is currently interested in the metabolic function and metabolic engineering of
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Hiroshi Ashihara is Professor of Metabolic Biology at Ochanomizu University in Tokyo. He obtained his PhD from the University of Tokyo in 1975, and did postdoctoral research at the University of Sheffield from 1977 to 1979. His research has focused on primary and secondary metabolism of nucleotides in plants and he has published more than 150 papers on purine, pyrimidine and pyridine nucleotide metabolism. He is currently interested in the metabolic function and metabolic engineering of nucleotide-related compounds. As experimental materials, in addition to model plants, he uses cell and tissue culture as well as intact plants of metabolically unique species including mangroves, coffee, cacao and special Chinese teas.
Hiroshi Sano obtained a PhD at the Biological Institute, Tohoku University, Japan. He then worked as a postdoctoral research associate at Freiburg University, Germany, and Dana-Farber Cancer Institute, Harvard Medical School, USA. In 1985, he was appointed as Professor of Plant Molecular Breeding at Akita Prefectural College of Agriculture, Japan. He moved to Nara Institute of Science and Technology in 1995. He has been interested in three different areas of plant science: epigenetics, plant–pathogen interactions and plant biotechnology. Professor Sano is also concerned about global environmental problems, and is involved in the molecular breeding of trees that are indispensable for the maintenance of the ecosystem. Currently he is a visiting professor at the Botanical Institute, Stockholm University, and serving as director of the Stockholm Office of the Japan Society for the Promotion of Science (JSPS).
Alan Crozier is a Botany graduate of the University of Durham. He obtained a PhD at Bedford College, University of London, carried out postdoctoral research at the University of Calgary and lectured at the University of Canterbury in New Zealand before joining the University of Glasgow in 1973 where he is currently Professor of Plant Biochemistry and Human Nutrition. He has published extensively on plant hormones and has been collaborating with Hiroshi Ashihara since 1994 on investigations into purine alkaloid biochemistry in coffee and teas. The activities of his research group are currently focussed on dietary flavonoids and phenolic compounds in fruits, vegetables and beverages, including teas and fruit juices, and their fate within the body following ingestion in relation to their potentially beneficial effects on health. Along with Hiroshi Ashihara, and Mike Clifford from the University of Surrey, he has recently co-edited a book entitled “Plant Secondary Metabolites: Occurrence, Structure and Role in the Human Diet” (Blackwell Publishing, Oxford).