Caffeine synthase

Purine alkaloids Caffeine synthase Camellia sinensis, Coffea arabica... 

Kato, M. Mizuno, K. Crozier, A. Fujimura, T. Ashihara, H. (2000) Caffeine synthase gaie from tea leaves. Nature, 406,956-7. 

Because the three genes encoding the canonical members of this family -SAMT, BAMT, and JMT have only recently been characterized, the full extent of this family is not known. Database searches using the BLAST 2.0 program reveal a number of unknown gene products and putative SAMT -like enzymes as well as caffeine synthases belonging to the type 3 family of MTs. Thus, the family includes not only carboxyl methyltransferases but also some alkaloid N-methyltransferases. 

The PA caffeine is produced from xanthosine via three distinct N-methylations (Fig.7.5).87 89 Partially purified enzyme extracts from tea (Camellia senensis) and coffee (Coffea arabica) were shown to exhibit all three activities, suggesting either that the A-methyltransferase steps in caffeine biosynthesis are catalyzed by a single enzyme, or by multiple enzymes with similar properties.90 However, a specific A-methyltransferase purified from coffee was active only toward 7-methylxanthine and theobromine91 An A-methyltransferase catalyzing the methylation of methylxanthines and designated caffeine synthase (CS) was purified from tea.92 CS catalyzes two consecutive methylations involved in the conversion of 7-methylxanthine to caffeine, but is inactive toward xanthosine, indicating that the first methylation proceeds via a different enzyme. Heterologous expression of the CS cDNA showed that the enzyme was active toward 7-methylxanthine, paraxanthine,... 

KATO, M, M1ZUNO, K, CROZIER, A., FUJIMURA, T., ASHIHARA, H., Caffeine synthase gene from tea leaves. Nature, 2000,406,956-957. 

Caffeine (Fig. 11.3), widely used by humans as a stimulatory drug, has so far been detected only in a few plant species. The biological roles of caffeine are believed to be in defense against herbivory or as an allelopathic response to potential competitors.83 Caffeine is derived from the purine alkaloid xanthosine. From xanthosine, three methylations are necessary to produce caffeine. First, xanthosine is methylated on N7 by 7-methylxanthosine synthase (MXS or 7NMT) to produce 7-methylxanthosine, which is enzymatically hydrolyzed to produce 7-methylxanthine and ribose.85,86 The methylations of N1 and N3 of 7-methylxanthine to produce 1,3,7-trimethylxanthine (caffeine) occur in young leaves of tea, and the same enzyme, caffeine synthase, apparently catalyzes both reactions.55 In coffee plants, caffeine is mainly found in the beans but also occurs in the leaves. Caffeine is stored in the vacuoles of coffee leaves as a complex with polyphenols such as chlorogenic acid.87 In contrast to tea, coffee plants appear to have separate enzymes for each step of N-methylation.57... 

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). 

Coffee and tea plants seem to contain a variety of N-methyltransferase enzymes that have varying substrate specificity (168, 169). For example, a caffeine synthase enzyme isolated from tea leaves catalyzes both the N-methylation of N-methylxanthine and theobromine (176). The substrate specificity of the methyltransferases can be changed by site-directed mutagenesis (177), and the crystal structure of two of the N-methyltransferases has been reported recently (178). 

Mizuno K, Kato M, Irino F, Yoneyama N, Fujimura T, Ashihara H. The first committed step reaction of caffeine biosynthesis 7-methylxanthosine synthase is closely homologous to caffeine synthase in coffee. FEES Lett. 2003 547 56-60. 

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.

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]... 

Various minor routes, dependent upon the broad specificities of the fV-methyltransferases, may also operate in addition to the main caffeine biosynthesis pathway. For instance, caffeine synthase catalyzes the biosynthesis of 3-methylxanthine from xanthine. Paraxanthine is biosynthesized from 7-methylxanthine. 3-Methylxanthine and paraxanthine are immediately converted into caffeine. Paraxanthine is the most active substrate of caffeine synthase, but only limited amounts of paraxanthine accumulate in plant tissues because the N-1 methylation of 7-methylxanthine is very slow [259, 260]. 

Fig. 8.8 Biosynthetic pathway for tropane and purine alkaloids, (a) Tiopane alkaloid ODC ornithine decarboxylase, PMT putrescine Ai-methyltransferase). (b) Purine alkaloid XMT xanthosine W-methyltransferase/7-methylxanthosine synthase, XN 7-methylxanthosine nucleotidase, MXMT 7-methylxanthine Ai-methyltransferase/theobromine synthase, DXMT dimethyl-xanthine Ai-methyltransferase/caffeine synthase) (Adopted from Ref. [15])...

Fig. 29.1 The biosynthetic pathways of caffeine from xanthosine. The major pathway that consists of four steps is shown with solid arrows. Conversion of 7-methylxanthosine to 7-methylxanthine is catalyzed by nucleosidase activity involved in 7-methylxanthosme synthase or a specific Af-methyl nncleosidase. Minor pathways, shown with dotted arrows, are theoretically possible. They may occur because of the broad substrate specificity of caffeine synthase or other Ai-methyltransferases. Enzymes 7mXS 7-methylxanthosine synthase, CS caffeine synthase, TS theobromine synthase, SAM 5-adenosyl-L-methionine, SAH S-adenosyl-L-homocysteine...

Kato M, Mizuno K, Fujimura T, Iwama M, Me M, Crozier A, Ashihara H (1999) PurificatiOTi and characterization of caffeine synthase from tea leaves. Plant Physiol 120 579-586... 

Kato M, Mizuno K, Crozier A, Fujimura T, Ashihara H (2000) A gene encoding caffeine synthase fi-om tea leaves. Nature 406 956-957... 

Figure 4.12 Biosynthesis of purine aikaioids. Enzyme abbreviations CS, caffeine synthase MXN, 7-methylxanthosine nucieosidase MXS, 7-methyixanthosine synthase.

Caffeine synthase, the majority of SAH hydrolase activity, and parts of the adenine-salvage pathway are localized to chloroplasts. In coffee SAM synthase is confined to the cytosol and SAM synthase genes fi-om tobacco and parsley lack a transit peptide. However, SAM synthase from tea is a chloroplastic enzyme, encoded by a nuclear gene (Koshiishi et al. 2001). The proposed model for the subcellular localization of caffeine biosynthesis begins with the production of homocysteine and its conversion to methionine in the chloroplasts. Methionine is then converted to SAM in the cytosol and transported back into the chloroplast to serve as the methyl donor in caffeine biosynthesis. Purine alkaloids are stored in vacuoles where they are thought to form complexes with chlorogenic acids (Mosli-Waldhauser and Baumann 1996). 

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