7-Methylxanthine Methyltransferase of Coffee Plants

Abstract

Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N. However, controversy exists as to the number and properties of the methyltransferases involved. Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants. Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves. Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT. It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring. K m values were estimated to be 50 and 12 M for 7-methylxanthine and S-adenosyl-L-methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine.

Cytology, biochemistry and molecular changes during coffee fruit development

“…For the caffeine pathway, ESTs homologous to 7-methylxanthosine synthase (CmXRS1), theobromine synthase (CTS1) and caffeine synthase (CCS1) were present in cDNA libraries from fruits (FR) as well as flower buds (FB) and leaf tissues (LV), confirming expression analyses performed by RT-PCR by others (Ogawa et al, 2001;Mizuno et al, 2003aMizuno et al, , 2003bUefuji et al, 2003). Interestingly, the ESTs coding for these enzymes were not over-expressed in cDNA libraries from plants submitted to biotic (RM, RX) or abiotic (SH) stress.…”

“…Several studies have been published dealing with gene cloning, expression analysis and protein studies of enzymes from the caffeine biosynthetic pathway (Moisyadi et al, 1998Ogawa et al, 2001;Mizuno et al, 2003a,b;Uefuji et al, 2003).…”

“…Due to the importance of caffeine in human health (Bättig, 1985;Schilter et al, 2001), caffeine metabolism has been studied for some time in coffee (see: Metabolism of alkaloids, Ashihara et al in this issue). Many genes implicated in its biosynthesis were recently cloned (Moisyadi et al, 1998Ogawa et al, 2001;Mizuno et al, 2003aMizuno et al, , 2003bUefuji et al, 2003) and the first trials to produce transgenic coffee plants with reduced caffeine content in beans are being carried out (Ogita et al, , 2004.…”

Biosynthesis and emission of insect-induced methyl salicylate and methyl benzoate from rice

“…In addition to SA and BA, other known carboxylic acid substrates of the SABATH family include jasmonic acid (JA) [34], indole-3-acetic acid [31,44e46], gibberellic acids [41], farnesoic acid [42], cinnamic acid and p-coumaric acid [16] and loganic acid [21]. Other members of the SABATH family are involved in caffeine biosynthesis by catalyzing nitrogen methylation [25]. Some SAMTs possess BAMT activity and vice versa.…”

Coffee biotechnology

“…N-methyltransferase proteins have also been purified (Mazzafera et al, 1994;Mösli Waldhauser et al, 1997;Moisyadi et al, 1998). Subsequently, cDNAs for 7-methylxanthine methyltransferase (MXMT or theobromine synthase) were successfully cloned from coffee plants (Ogawa et al, 2001;Mizuno et al, 2003).…”