Separation of the N-7 methyltransferase, the key enzyme in caffeine biosynthesis.
Caffeine biosynthesis comprises sequential methylations at N-7, N-3 and N-1 of the xanthine ring catalysed by S-adenosyl-L-methionine (SAM)-dependent methyltransferase activities that, to date, have not been resolved. Enzyme extracts were prepared from young, emerging coffee leaflets and following anion exchange chromatography, chromatofocusing facilitated the clear separation of the N-7-methyltransferase from the N-3- and N-1-methyltransferase activities. All three N-methyltransferases co-eluted when analysed by gel filtration chromatography and their native molecular mass was ca 67 kDa. Photoaffinity labelling with [methyl-3H]SAM followed by SDS-PAGE of a chromatofocusing-purified preparation containing only N-7-methyltransferase activity demonstrated the presence of a single labelled band of 40 kDa. Similar analysis of a gel filtration purified preparation containing all three N-methyltransferase activities revealed the presence of three labelled bands at 49, 43 and 40 kDa. It remains to be determined whether the 49 and 43 kDa bands are associated with the N-3 and N-1-methyltransferases or whether they are unrelated SAM-dependent methyltransferases or other SAM-binding proteins.