Theobromine biosynthesis

The methylxanthines of interest are caffeine (1,3,7-trimethylxanthine), theophylline (1,3-dimethylxanthine), and theobromine (3,7-dimethylxanthine) and they occur in coffee, tea, mate, cocoa products, and cola beverages. This chapter is an introduction to their chemistry, isolation, and biosynthesis. While the class of methylxanthines is large and comprised of more members than these three, this chapter will essentially be limited to caffeine, theobromine, and theophylline. 

Several studies have investigated the biosynthesis of caffeine in tea. The results of a study by Suzuki and Takahashi27 30 suggest a pathway for caffeine biosynthesis in tea from 7-methylxanthine to theobromine and then to caffeine. Additionally they suggest that theophylline is synthesized from 1-methylxanthine. Another study by Ogutuga and Northcote31 proposes a pathway through 7-methylxanthosine to theobromine followed by caffeine. 

While caffeine biosynthesis in coffee and tea has been reasonably well investigated, little information is available about the biosynthetic pathways of methylxanthines in cacao. Published studies34 35 have established the presence of 7-methylxanthine and adenine in cocoa. Since both coffee and tea exhibit similar pathways where theobromine is a direct precursor for caffeine, it is reasonable to assume that a similar mechanism is possible in cacao. 

Ogita, S, Uefuji, H Morimoto, M. and Sano, H. 2004. Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties. Plant Molecular Biology, 54(6) 931-941. 

Several purine derivatives are found in nature, e.g. xanthine, hypoxanthine and uric acid. The pharmacologically important (CNS-stimulant) xanthine alkaloids, e.g. caffeine, theobromine and theophylline, are found in tea leaves, coffee beans and coco. The actual biosynthesis of purines involves construction of a pyrimidine ring onto a pre-formed imidazole system. 

Figure 2.17 Biosynthesis of theobromine and caffeine in Coffea arabica. SAM, 5-adenosyl-L-methionine SAH, 5-adenosyl-L-homocysteine.

Figure 4 Caffeine biosynthesis. XMT, xanthosine N-methyltransferase (also called 7-methylxanthosine synthase) XN, methyixanthosine nucleotidase MXMT, 7-methylxanthine-N-methyltransferase (also called theobromine synthase) DXMT, dimethyIxanthine-N-methyltransferase (also called caffeine synthase).

The biosynthesis of caffeine begins with the methylation of xanthosine to yield N-methylxanthosine by the enzyme xanthosine N-methyltransferase (XMT) (also called 7-methy-Ixanthosine synthase) (171-173). N-methylxanthosine is converted to N-methylxanthine by methylxanthine nucleosidase, an enzyme that has not been cloned yet (174). N-methylxanthine is converted to theobromine by 7-methylxanthine-N-methyl-transferase (MXMT) (also called theobromine synthase), a second N-methyltransferase (171, 175). Theobromine is converted to caffeine by a final N-methyltransferase, dimethylxanthine-N-methyltransferase (DXMT) (also called caffeine synthase) (171). 

Scheme 4. Biosynthesis of the purine alkaloids caffeine and theobromine. Molecular clones have been isolated for all enzymes shown. Abbreviations CS, caffeine synthase MXN, 7-methyIxanthosine nucleosidase MXS, 7-methyIxanthosine synthase.

The biosynthesis of caffeine takes the same path up to xanthosine-5 -phosphate, which is here dephosphorylated and methylated at N-7. However, there are also reports that xanthosine-5 -phosphate can be methylated directly. [506] Afterthe sugar residue is cleaved off, two additional methylation steps take place via the intermediates 7-methykanthine and theobromine. [507]... 

Most recently, genetic engineering opened the opportunity to knock-out caffeine-synthase and eliminate both of the last steps in the biosynthesis, methyla-tion of 7-methylxanthine and theobromine respectively. The goal is to cultivate thereby caffeine-free tea and coffee, without affecting their high polyphenol content as current decaffeination processes do. [511, 512]... 

The biosynthesis pathway of theobromine and caffeine has been the subject of many studies over the years. Caffeine is produced from the purine nucleotides AMP, GMP, and/or IMP, and theobromine is the immediate precursor of caffeine. 

The biosynthesis of caffeine was investigated through incorporation experiments using the stems of Cqffea and Camellia plants [17]. As a result, it became clear that methylation of the N-7 position occurred on xanthosine derived from XMP, which originated from AMP via IMP. Caffeine is then biosynthesized through 7-methyl xanthosine, 7-methyl xanthine, and theobromine, as shown in the figure. 

Camellia (Thea) sinensis (tea) and Cqffea arabica (coffee) plants. Caffeine was formed rapidly by extracts of green coffee berries, but little by more mature ones, and not at all by seedlings. Biosynthesis of caffein proceeds from 7-methylxanthosine in the presence of an active purine nucleoside phosphorylase or 7-methyl-A -nucleoside hydrolase. Methionine and 5-adenosylmethionine serve as precursors for the methyl groups of purine alkaloids. These act in the presence of methyltransferases on 7-methylxanthine (38) and theobromine (31) to produce caffeine. A pathway for the origin of these compounds in coffee and tea plants has been proposed (Suzuki et al., 1992 Waller and Dermer, 1981) (Fig. 37.10). 

Caffeine constitutes 2.5-5.5% of the dry matter of tea leaves. It is of importance for the taste of tea. Theobromine (0.07-0.17%) and theophylline (0.002-0.013%) are also preset but in very low amounts. The biosynthesis of these two compounds involves methylation of hypoxanthine or xanthine ... 

The biosynthesis of caffeine begins with xanthosine, which is converted into 7-methylxanthosine, 7-methylxanthine, theobromine and caffeine, in that order. The genes encoding three distinct A/ methyltransferase enzymes used to make caffeine from xanthosine have been isolated and then expressed in tobacco plants. The resulting caffeine-containing tobacco plants are unpalatable to tobacco cutworms (Spodoptera liturd). 

Zheng, X.-Q., Koyama, Y., Nagai, C. and Ashihara, H. (2004) Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits. /. Plant Physiol, 161, 363-369. 

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