ISHS Acta Horticulturae 461: International Symposium on Biotechnology of Tropical and Subtropical Species Part 2
CLONING AND CHARACTERIZATION OF A CDNA ENCODING XANTHOSINE-N7-METHYLTRANSFERASE FROM COFFEE (COFFEA ARABICA) |
| Authors: | S. Moisyadi, K.R. Neupane, J.I. Stiles |
| Keywords: | Coffee, caffeine biosynthesis, methyltransferase |
| Abstract:
Xanthosine-N7-methyltransferase (XMT) is the first enzyme unique to the caffeine biosynthetic pathway in coffee, tea and perhaps other caffeine-containing plants. This enzyme methylates xanthosine specifically at the N7 position. XMT does not methylate at other positions on the xanthine ring and does not methylate other purines or related compounds. Using standard protein purification techniques this enzyme was purified and a partial amino acid sequence was obtained from several fragments of a tryptic digest. Synthetic oligonucleotides were prepared based on the amino acid sequences and used to amplify a portion of the coding sequence using mRNA from young leaves and the reverse transcriptase polymerase chain reaction (PCR). The PCR-generated fragment was used to screen a cDNA library constructed from mRNA isolated form young leaves. Several cDNAs were isolated and the largest was completely sequenced. This cDNA encodes a protein of 371 amino acids (41 kD) with a calculated pI of 5.8. These values are nearly identical to the molecular weight of 37.6 kD determined by SDS-gel electrophoresis and the pI of 5.36 determined from isoelectric focusing. XMT does not show significant homology to other known proteins. This cDNA is currently being used to alter caffeine content in coffee by antisense and co-suppression. | |
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