7-Methylxanthine methyltransferase of coffee plants. Gene isolation and enzymatic properties

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

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    101 Citations (Scopus)

    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. Km 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.

    Original languageEnglish
    Pages (from-to)8213-8218
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume276
    Issue number11
    DOIs
    Publication statusPublished - 2001 Mar 16

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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