Over-expression of the yeast multifunctional arom protein.

The pentafunctional arom protein of Saccharomyces cerevisiae is encoded by the ARO1 gene. Substantial elevation of the levels of the arom protein (25-fold) was achieved in yeast using a vector that exploited the ubiquitin-fusion cleavage system of yeast. However, attempts to express the N-terminal 3-dehydroquinate synthase domain (E1) or the internal 3-dehydroquinase domain (E2) using the same system did not succeed. The yeast arom protein was successfully purified from the over-expressing transformant, and was found to possess all five enzymatic activities in a ratio similar to that observed in crude cell extracts. The purified material consisted mainly of a polypeptide that co-migrated in SDS-PAGE with intact arom proteins from other species.

Biosynthesis of Caffeine in Leaves of Coffee.

The levels of endogenous caffeine and theobromine were much higher in buds and young leaves of Coffea arabica L. cv Kent than in fully developed leaves. Biosynthesis of caffeine from 14C-labeled adenine, guanine, xanthosine, and theobromine was observed, whereas other studies (H. Ashihara, A.M. Monteiro, T. Moritz, F.M. Gillies, A. Crozier [1996] Planta 198: 334-339) have indicated that there is no detectable incorporation of label into caffeine when theophylline and xanthine are used as substrates for in vivo feeds with leaves of C. arabica. The capacity for caffeine biosynthesis, especially from guanine and xanthosine, was reduced markedly in both fully developed mature and aged leaves. Data obtained in pulse-chase experiments with young leaves indicate the operation of an AMP -> IMP -> xanthosine 5[prime]-monophosphate (or GMP -> guanosine) -> xanthosine -> 7-methylxanthosine -> 7-methylxanthine -> theobromine -> caffeine pathway. The data obtained provide strong evidence against proposals by G.M. Nazario and C.J. Lovatt ([1993] Plant Physiol 103: 1203-1210) concerning the independence of caffeine and theobromine biosynthesis pathways and the role of xanthine as a key intermediate in caffeine biosynthesis.

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.