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J. Biol. Chem., Vol. 276, Issue 11, 8213-8218, March 16, 2001

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7-Methylxanthine Methyltransferase of Coffee Plants
GENE ISOLATION AND ENZYMATIC PROPERTIES^*

Mikihiro Ogawa, Yuka Herai, Nozomu Koizumi, Tomonobu Kusano, and Hiroshi Sano

From the Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara 630-0101, Japan

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 µM 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.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession numbers AB039725 (CaMTL1), AB048792 (CaMTL2), AB048793 (CaMTL3), and AB048794 (CaMXMT).

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