Caffeine biosynthesis starts with the metabolically channelled formation of 7-methyl-XMP--a new hypothesis

Young coffee leaflets were fed with [U-14C]adenine of [Me-14C]methionine, and the incorporation of radioactivity into purines (purine bases, nucleosides, nucleotides, nucleic acids) and purine alkaloids (PA) (7-methylxanthine, theobromine, caffeine) was determined after 4 and 24 hr. Labelling of PA was considerable after 4 hr (i.e. about 38 or 71% of the recovered radioactivity from [U-14C]adenine and [Me-14C]methionine, respectively) and, on an absolute basis, distinctly higher after 24 hr signifying a continuous synthesis and precursor supply in the isolated leaflets. Despite these favourable conditions and the most selective approach to methyl group labelling, i.e. use of [Me-14C] methionine, we were not able to show the incorporation of radioactivity into either 7-methylxanthosine, the formerly postulated key compound at the onset of PA synthesis, or any monomethylated purine other than 7-methylxanthine. The same negative result was obtained in our recent studies with coffee cell suspension cultures and implies that the first methylation step in caffeine synthesis is metabolically channelled. Additionally, enzyme extracts were prepared from the same tissue and methyltransferase activity was measured using various substrates. Among them only xanthosine and XMP were methylated, and exclusively at the N-7 position of the purine ring. XMP N-methyltransferase activity, which had not been detected before, was measured under conditions which strongly reduced hydrolysis of both educt and product (e.g. by Na2MO4 as inhibitor of nucleotidase activities). These results, together with our recent studies, allow us to present a nova hypothesis regarding caffeine biosynthesis which favours methylation of XMP rather than xanthosine as the crucial step leading from primary metabolism into caffeine synthesis. However, this first methylation is considered to be metabolically channelled.